Ligand-mediated immunofunctional hormone binding protein assay method

ABSTRACT

A ligand-mediated immunofunctional assay (LIFA) method for detecting the presence and the concentration of polypeptide hormone binding proteins which comprises capturing the binding protein with a solid phase bound first antibody, saturating the bound hormone binding protein with the ligand polypeptide hormone, and detecting the bound ligand polypeptide hormone with a detectably labeled second antibody specific for the ligand polypeptide hormone. In the absence of added saturating polypeptide hormone, the LIFA measures the amount of hormone binding protein bound to the endogenous ligand polypeptide hormone. A growth hormone binding protein assay illustrates the method of the present invention. LIFA assay results indicate that increased binding protein substantially increases growth hormone activity. Methods of use and formulations of growth hormone binding protein, growth hormone, insulin-like growth factor-I and insulin-like growth factor binding protein are disclosed.

This is a continuation of applications Ser. No. 08/408,094, filed onMar. 21, 1995, which is a continuation of Ser. No. 08/039,093, filed onApr. 9, 1993, now abandoned, which is a 35 USC §371 of PCT/US91/08664,filed on Nov. 19, 1991which is a continuation-in-part of Ser. No.07/615,538, filed on Nov. 19, 1991, now issued as U.S. Pat. No.5,210,017, which applications are incorporated herein by reference andto which application priority is claimed under 35 USC §120.

BACKGROUND OF THE INVENTION

1. Field of the Invention

A novel ligand-mediated immunofunctional assay (LIFA) method isdescribed for detecting the presence and quantitating the amount of apolypeptide hormone binding protein in a biological fluid and/ordetermining the amount of the ligand polypeptide hormone specificallybound to the hormone binding protein. This modified immunometric assayfor a hormone binding protein uses: 1) a first solid phase boundantibody to capture the hormone binding protein; 2) a saturating amountof ligand hormone; and, 3) a labeled second antibody specific for theligand hormone, The LIFA method is exemplified by a growth hormonebinding protein assay.

2. Description of the Background Art

A hormone binding protein (HBP) is a carrier protein found in biologicalfluids which has binding specificity for a ligand polypeptide hormone.Examples of such HBP's are growth hormone binding protein (GHBP),epidermal growth factor (EGF) binding protein, insulin-like growthfactor 1 and 2 (IGF-1, IGF-2) binding proteins (six of them), plateletderived growth factor (PDGF) binding protein, nerve growth factor (NGF)binding protein, insulin binding protein, corticotropin releasing factor(CRF) biding protein, transforming growth factor beta (TGF-β) bindingprotein and activin binding protein (Follistatin).

One of the best characterized polypeptide hormone binding proteins isthe GHBP. The GHBP discussed in this invention is the extracellulardomain of the GH receptor which circulates in blood and functions as aGHBP in several species (Ymer, S. I., and Herington, A. C., Mol. Cell.Endocrinol. 41:153 [1985]; Smith, W. C. and Talamantes, F.,Endocrinology 123:1489-94 [1988]; Emtner, M., and Roos, P., ActaEndocrinologica [Copenhagen] 122:296-302 [1990], including man (Baumann,G. et. al., J. Clin. Endocrinol. Metab. 62:134-141 [1986]; Herington, A.C. et al., J. Clin. Invest. 77:1817-1823 [1986]). Little is known aboutthe fate of the GHBP or its regulation in various physiological andpathological conditions.

Hormone binding proteins have been assayed by antibody basedprecipitation methods where the ligand is labeled and the antibody isspecific for the binding protein itself. Monoclonal antibodies specificfor human growth hormone binding protein (hGHBP) were used by Barnard etal., J. Endocrinol, 123(2):327-32[1989]; for rabbit GHBP by Ymer et al.,Endocrinology, 125(2):993-9[1989]; and mouse by Smith et al.,Endocrinology 123(3):1489-94[1988]. Currently available methods forestimating GHBP levels in blood are based on incubation of the samplewith radiolabeled GH, followed by separation of bound and free GH(Baumann, G. et al., Acta Endocrinologica (Copenhagen) 119: 529-34[1988]; Amit, T. et al., J. Clin. Endo Metab. 71:474-479 [1990]). Theresults obtained by these assays are difficult to interpret due tointerference by endogenous GH (Baumann, G. et al., J. Clin. Endocrinol.Metab. 62:134-141 [1986]). Others who have used labeled growth hormoneto detect GHBP are: Emtner et al., Acta Endocrinol 122(3):296-302,(1990); Silbergeld et al., Clin. Endocrinol. 31(3):295-303 (1989);Daughaday et al., J. Clin. Endocrinol Metab., 65(5):1072-4, (1987);Herington et al., J. Clin. Invest. 77(6):1817-23, (1986); and Laron etal., Acta Endocrinol. 121(4):603-8 (1989). These assays for GHBP inblood have serious problems. They are laborious, requiring separation ofcomplexed GHBP-GH by size-exclusion chromatography or antibodyprecipitation, and they may not give consistent results from onelaboratory to another. In addition, they generate results that arearbitrary (i.e. not calibrated to a common protein standard) andinfluenced by endogenous growth hormone. Therefore, there is a need foran improved assay method which will allow detection of all thepolypeptide hormone binding proteins, including those bound toendogenous polypeptide hormone.

Methods for the production of monoclonal antibody-producing hybridomasare disclosed by Kipps and Herzenberg in Clinical Endocrinology andMetabolism, Vol 62, No. 1, page 108.1-108.9 (1986)

A monoclonal antibody-based immunoradiometric assay for IGF bindingprotein was described by Pekonen et al, J. Immunoassay 10:325-37 (1989).Immunometric or sandwich immunoassays using high affinity monoclonalantibodies were taught in David et al., U.S. Pat. No. 4,486,530. Suchsandwich assays have an antigen with two or more epitopes sandwichedbetween two antibodies. Circulating proteins that bind non-polypeptidehormone ligands, such as the thyroxine binding protein, have beenmeasured using fluorescent labeled tracer (U.S. Pat. No. 4,476,228) inorder to determine the number of binding protein sites not occupied bythyroxine. Iodothyronine immunoassays in a biological fluid usingblocking agents and thyroxine binding globulin were described in Gordonet al, U.S. Pat. No. 4,622,293.

Specific binding pairs (SBP) are discussed in reference toantigen-antibody reactions, ligand-receptor, hormone-receptor andlectin-oligosaccharide (U.S. Pat. No. 4,956,302). Zuk et al., (U.S. Pat.No. 4,594,327) describes assays of whole blood to detect members of suchSBPs wherein one member of the SBP must be attached to the solid phaseprior to contacting the blood. The other second member of the SBP isdetected using labeled second SBP member in competitive reactions.Similarly, Weng et al. (U.S. Pat. No. 4,737,456) describes a method ofreducing interfering substances in assays of a SBP member wherein theindividual member of the SBP is labeled. The receptor is used in acompetitive assay to capture both labeled and unlabeled ligand, not toanalyze for the presence of and quantify receptor as in the presentinvention. A method for the determination of an antigen using twoantibodies is disclosed in U.S. Pat. No. 4,343,896.

Problems in Previous Polypeptide Hormone Binding Protein Assays

Problems in detecting HBP can be best illustrated by the problemsencountered in detecting GHBP. Previous methods for determining thepresence of GHBP in biological fluids were not as accurate as desiredand frequently required the use of radioactive materials. The presentinvention avoids the problems of the previous assay methods in that it(a) does not require the use of radiolabeled GH; (b) does not requirethe removal of endogenous GH from the GHBP; (c) does not require anyform of size separation of GH from the GH-GHBP complex; and, (d)measures the actual mass or absolute amount of GHBP rather than arelative amount reported in arbitrary units. The present invention hasthe added advantage that it measures the binding capacity of thecirculating GHBP and is able to measure the degree of saturation of theGHBP with respect to GH. Moreover, the present invention is specific forGHBP by substantially reducing background assay noise that causesimprecision. The assay of the present invention avoids the problems instandard immuno-metric assays by using a first antibody to capture theGHBP and a second detectably labeled antibody to measure the present ofbound GH. The use of a second antibody specific for another epitope onthe GHBP would not determine whether the GHBP was functional, and inaddition, it could increase the background due to other serum proteinswhich bound both antibodies.

In order to study the function of the endocrine system it is essentialto have access to reliable methods for quantitation of all parts of thesystem, i.e. the hormone, its being protein and the hormone-bindingprotein complex. These measurements have not been previously achievedbecause of interference in the assays by the different components andthe fact that both the hormone and the BP can be present in free andcomplexed forms. It is even more complicated when there are severaldifferent binding proteins for the same hormone, as for the IGF-1system. However, the present invention teaches how the use of monoclonalantibodies directed at a specific binding protein can measure the amountand degree of saturation of that specific binding protein. In the caseof GH, for which a second binding protein with lower affinity has beendescribed (Baumann, G. and Shaw, M. A., J. Clin. Endocrinol. Metab.70:680-686 [1990]), this binding protein appears to be structurallyunrelated to the GH receptor and should not be detected in our assay.However, this other GH binding protein may also be detected in thepresent invention's assay method once the other binding protein isisolated and appropriate antibodies are raised.

Currently, the standard method for quantitation of carrier proteins forpeptide hormones is incubation of serum with the radiolabeled ligand,followed by chromatography or precipitation to separate the bound andfree hormone. These procedures are laborious and the results oftendifficult to interpret because of the interference by endogenous hormonein the sample. These assay methods give an estimate of the bindingcapacity of serum proteins of a certain size but the activities ofdifferent binding proteins of similar size are not distinguished and therelative proportion of free and complexed BP cannot be determined.Another disadvantage is that the results are expressed in relation toreference serum pools, which makes it difficult to compare the resultsin different studies. In the present invention, we developed an assayfor the GHBP by choosing a new approach. This assay was surprisinglyable to precisely detect individual hormone binding proteins in a waythat has not been previously demonstrated.

The LIFA, which is the preferred method used in the present invention,is simple to use and has the advantage that only functional bindingprotein is detected. When the assay method is applied to GHBP, bothtotal and endogenously complexed GHBP are measured, and the assay doesnot require removal of endogenous GH from the GHBP or procedures toseparate free GH from the GHBP complex. In contrast to previous methods,endogenous GH does not interfere in the assay; instead, bound GH, eitherendogenous or exogenously added, is used to detect the total andcomplexed GHBP. In fact, one cannot use the GH as the first member boundto the solid phase since GH cannot complex with binding protein that isalready complexed with endogenous GH. Therefore, one requirement of thepresent assay method is for a solid phase coat antibody which recognizesthe binding protein both in a free and complexed form. The assay methodtaught in this invention can also be used to measure the total bindingcapacity and the saturation of other polypeptide hormone-bindingproteins with respect to the ligands that they bind.

Therefore, the present invention describes the development of a novel,sensitive and specific enzyme-linked immunosorbent assay (ELISA) forquantitation of biologically active HBP in biological fluids. The assaycan also be used to measure the concentration of the ligand-hormonebinding protein complex. The method of the present invention provides anumber of advantages relating to ease of analysis, sensitivity,precision and reliability which will be more apparent as the details ofthe method are discussed.

SUMMARY OF THE INVENTION

A LIFA method is described for detecting the present and theconcentration of a polypeptide hormone binding protein and/or the degreeof saturation of the hormone binding protein with its specific ligandhormone. Functional binding between the hormone binding protein and theligand hormone is required in the assay. As illustrated in FIG. 1, steps1-3, a monoclonal or polyclonal antibody which binds one or more hormonebinding protein epitopes, which are exposed in both the free and theligand hormone-associated binding protein, is used to capture thehormone binding protein on a solid matrix. In step 3, the capturedhormone binding protein is incubated with the ligand hormone to saturatethe hormone binding protein sites specific for the ligand hormone. Inone option, the hormone binding protein is not saturated with the ligandhormone prior to the step 4 incubation with hormone specific labeledantibody. This allows determination of the level of endogenous ligandhormone associated with the hormone binding protein prior to incubationwith added exogenous ligand hormone. The hormone binding protein toincubation with added exogenous ligand hormone. The hormone bindingprotein and the ligand hormone may be simultaneously incubated togetherand with the solid phase capture antibody or they may be sequentiallyincubated with the capture antibody. In step 4, a detectably labeledmonoclonal or polyclonal antibody, that binds to one or more epitopes onthe ligand hormone at a site that is different from where the hormonebinding protein binds, stably binds to the ligand hormone-hormonebinding protein complex. When the hormone binding protein is saturatedwith added hormone (step 3) the total amount of detected hormone is ameasure of the amount of binding protein present. When the hormonebinding protein is not saturated with added hormone, the amount ofdetected hormone is a measure of endogenous ligand hormone associatedwith the hormone binding protein. A comparison of the two values allowsa determination of both the amount of hormone binding protein presentand the relative saturation with endogenous ligand hormone.

The therapeutic use of GHBP alone and in combination with GH tostimulate growth hormone responsive tissues is shown. The use of aGHBP-GH therapeutic composition is shown to result in greaterstimulation of GH responsive tissues with the use of less GH.Furthermore, the GHBP-GH composition is longer lasting followingadministration thereby permitting less frequent administration than withGH alone.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic of Assay Procedure for detecting human growth hormonebinding protein.

FIG. 2. Coat MAb selection. % bound GH-I¹²⁵ by MAb 5, 43 and 263 wereploted vs the three different incubation configurations. The immobilizedMAbs were evaluated in three different experiments. In the firstexperiment, using sequential incubation steps, GHBP was first incubatedwith MAb coated on the well, followed by addition of radiolabelled GH(GH-I¹²⁵). In the second experiment the reaction of GHBP and GH-I¹²⁵ wascarried out simultaneously in MAb coated wells. In the third experiment,GHBP was pre-incubated with hGH-I¹²⁵ overnight at 4° C. and then addedto the MAb coated well.

FIG. 3. Comparison of two standard curves generated with and withoutpreincubation with GH. One set of standards were incubated with GH(final concentration 200 ng/ml) over night at +4° C., the controlstandards were incubated with assay buffer. The samples so generatedwere then assayed in the LIFA.

FIG. 4. Standard curves of hGHBP using 22 kD hGH as ligand, hGHBP using20 kD as ligand, and hGHBP using a combination of hGH 22 kD and 20 kD.

FIG. 5. Plot of theoretical hGHBP concentration based on dilution ofthree different serum samples vs. hGHBP concentration in the serumsamples diluted in assay buffer and measured by LIFA.

FIG. 6. Total- and hGH-bound- hGHBP levels in random serum samples from16 healthy adults (sample #1-16) and two patients with Laron typedwarfism (sample #17 and 18).

FIG. 7A-7B. Cross correlation of GH and GHBP (7A) Twenty-four hourplasma profiles of GH (top panel), GH/GHBP-complex (middle panel) andtotal GHBP concentration (bottom panel) in samples from a a 15 year oldboy (profile No 15, Table 4); (7B) statistical cross correlationanalysis of data from (A).

FIG. 8A-8E. GH binding protein levels in National Cooperative GrowthStudy (NCGS) patients indicating the log concentration of GHBP vs theage of patient. The crossbars represent mean values; solid verticallines are plus or minus 1 SDs; dotted vertical lines are plus or minustwo standard deviations (SDs). The separate black dots each representone patient. (8A) Idiopathic growth hormone deficiency (GHD) for males;(8B) Idiopathic GHD for females; (8C) Idiopathic short stature (ISS) formales; (8D) ISS for females; (8E) Turner Syndrome.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Polypeptide Hormone Binding Protein Assay

Assaying for the presence of polypeptide hormone binding protein uses:(1) a first capture monoclonal antibody specific for the bindingprotein; (2) ligand polypeptide hormone and (3) a second monoclonalantibody specific for the ligand polypeptide hormone. The LIFA we havedeveloped is a simple and sensitive method which allows quantitation ofthe total concentration of functional hormone binding protein in asolution. The assay's binding protein detection range may vary fromabout 4 to 20,000 pmol/L, more preferably from about 10 to 4000 pmol/L,and most preferably from about 20 to 2000 pmol/L. The assay can also bemodified to measure the concentration of the circulating ligandhormone-binding protein complex. A monoclonal antibody directed againstthe binding protein, which recognizes both free and hormone boundbinding protein is used to capture the binding protein on a solid phase,such as a microtiter plate. The samples are incubated with the ligandhormone to saturate all binding sites and an anti-hormone antibody isused to detect the amount of hormone (endogenous and exogenous) whichhas bound to the binding protein. The same procedure, but withoutincubation with ligand hormone, allows quantitation of the levels ofcirculating hormone-binding protein complex.

This LIFA may be used for any polypeptide hormone binding protein. Itoffers the ability to accurately detect the concentration of functionalbinding protein present in any biological fluid and to measure theendogenous level of saturation of functional binding protein with itsligand polypeptide hormone. For the first time this assay method permitsthe measurement of functional HBP and the calibration of a HBP assay toa verifiable mass unit as opposed to serum units, etc. While this can beaccomplished by using naturally produced HBP and ligand hormone (ifavailable), it is usually accomplished by conducting the assay withrecombinant polypeptide hormones and creating a standard curve basedupon recombinant HBP. Surprisingly, the present LIFA method may be usedto detect both the total HBP and the amount of HBP complexed withendogenous polypeptide hormone. Therefore, for the first time there isan accurate method of determining the amount of functional HBP which isnot influenced by ambient polypeptide hormone.

In one modification of the present immunofunctional HBP assay method,the added ligand hormone contains a second detectable marker which doesnot hinder the binding of the detection antibody. This second marker onthe ligand hormone and the first marker on the detection antibody arenot the same. By separately measuring the amount of added ligand hormonebound and the amount of bound antibody, the percent saturation of theHBP with ligand hormone and one on the detection antibody, permits asimultaneous determination of the amount of bound exogenous ligandhormone and total HBP.

The assay of the present invention may also be used to quantitate theamount of ligand hormone in an unknown sample. Recombinant HBP, or HBPstripped of endogenous ligand hormone, is bound to the solid phase as inthe assay method. First, the unknown sample containing the ligandhormone is contacted with the antibody bound HBP. Second, the ligandhormone specific labeled antibody is added and the amount bound isdetermined. This application of the LIFA permits the use of HBP insmaller amounts than would be used by directly coupling the HBP to thesolid phase. The use of antibody to bind the HBP assures that theorientation is such that the HBP binding sites for the ligand hormoneare not blocked.

Human Growth Hormone Binding Protein Assay

Assaying for the presence of hGHBP uses: 1) a first capture monoclonalantibody specific for the binding protein; 2) growth hormone; and 3) asecond monoclonal antibody specific for growth hormone (FIG. 1). TheLIFA we have developed is a simple and sensitive method which allowsquantitation of the total concentration of functional growth hormonebinding protein in biological fluids. The assay can also be modified tomeasure the concentration of the circulating ligand-binding proteincomplex. A monoclonal antibody directed against the growthhormone-binding protein, which recognizes both free and GH bound bindingprotein is used to capture the binding protein on a microtiter plate(FIG. 1, step 1). The samples are incubated with human growth hormone tosaturate all binding sites (FIG. 1, step 3) and an anti-hGH antibody isused to detect the amount of hGH (FIG. 1, step 4) (endogenous andexogenous) which has bound to the binding protein. The same procedure,but without incubation with added hGH, allows quantitation of serumlevels of circulating hGH-binding protein complex.

A preferred assay herein is a monoclonal antibody (MAb)-based sandwichELISA for quantitation of GHBP in biological fluids such as human serumor plasma. The assay, which only detects functional GHBP, can be used tomeasure both total and complexed GHBP, and the degree of saturation ofthe GHBP with its ligand can therefore be calculated. In an assaydeveloped for hGHBP, the 263 (coat) MAb binds free GHBP as well as GHBPbound to hGH, and is used to capture the GHBP on microtiter plates. Theconjugated MAb MCB, which is directed against hGH, is used to detect thehGH that is bound to the immobilized GHBP. The total amount of GHBP ismeasured by saturating the binding sites with hGH, followed by detectionof the total (endogenous and exogenous GH) bound to the GHBP. Theconcentration of the endogenous GH-GHBP complex is obtained byincubating the samples with the conjugated antibody without previoussaturation of the GHBP with GH. The assay is sensitive and appears tocover the physiological range since random samples from 16 normal adultsall had clearly detectable GHBP levels. Spike recovery experiments showthat the assay is useful for measuring GHBP levels in both serum andplasma, with recoveries ranging from 89.1 to 113.6%.

One requirement of the present assay method is for a coat antibody whichrecognizes the antigen both in a free and complexed form, which forhGHBP is the MAb 263. Using the techniques described herein forgenerating such antibodies, we believe that the principle behind ourGHBP LIFA could be used to measure the total binding capacity and thesaturation of other polypeptide hormone-binding proteins. The differencein % bound observed when MAb 263 was tested in three different assayformats (FIG. 2 is due to the assay kinetics rather than the MAb bindingcharacteristics. In the overnight pre-incubation experiment, the GHBPhad the longest incubation with GH-I¹²⁵ and hence the highest % bound.Even though the reaction time (4 h) was the same in the simultaneous andthe sequential assay, the complexing of GH-I¹²⁵ to GHBP would be fasterfor a homogeneous system (simultaneous) vs. a heterogeneous system(sequential), thus the % bound would be higher.

The GH used to saturate the GHBP may be a GH analog wherein there is abinding site specific for the GHBP and one or more exposed epitopes forbinding the GH specific antibody.

Applications of the GHBP Assay

The results of the assay for GHBP and the degree of saturation of theGHBP with GH may be used for the diagnosis and treatment of growthdeficiency. This assay is anticipated to provide an integrated index ofthe exposure of an organism to GH. It provides a more precisedetermination of total and free GH, which will better identify patientswho are relatively GH-deficient and will benefit from GH replacementtherapy. In general, the assay would be of value in any situation whereGH is administered therapeutically for treating a clinical condition orwhere GH is present in excessive amounts and involved in causing orreflecting a clinical pathophysiology. The results provide an improvedmethod for assessing compliance with GH replacement therapy andtitrating dose and individualizing GH therapy for individual patients orthose with particular clinical manifestations of GH deficiency orexcess.

Use of these assay results facilitates diagnosing resistance to GHaction due to decreased amounts or decreased binding activity of theGHBP or of the GH receptor, such as in hereditary or acquired syndromes.Hereditary syndromes, including Laron dwarfism and other syndromes ofshort stature such as ideopathic short stature are revealed whenabnormalities of the GHBP are shown to exist. The invention also assistsin the identification and treatment of acquired syndromes includingdisease states where there is diminished or excessive expression ofGHBP, such as in liver disease. Other examples of the use of the presentassay invention include detection of diseases of ovarian function,diseases of joints or bone, or hypothalamic or pituitary diseasedcausing excess GH or GHBP production. Clinical use of GHBP, or theGHBP/BH complexes as a therapeutic agent, are aided by the presentinvention because the measurement of GHBP or GHBP+GH after theiradministration assists clinical practice by allowing a determination ofthe present of therapeutically effective or therapeutically ineffectiveamounts in bodily fluids.

All parts of the growth promoting system are interrelated and it isexpected that administering growth hormone releasing factor, growthhormone inhibitory factor (somatostatin), IGF-1, or IGF-1 bindingproteins, may perturb GHBP concentrations. The measurement of GHBPassists clinical practice in assessing the suitability of patients fortreatment with these proteins and the efficacy of such treatments.

Clinical Applications of GHBP Assay

Progress in our understanding of GHBP requires a quick, convenient andaccurate assay for GHBP. The initial paper describing a GHBP waspublished in 1964 (Hadden et al., Nature 202:1342 [1964]). The serumGH-binding protein was better characterized in the mouse in 1977(Peeters, S. and Friesen, H. G., Endocrinology 101:1164 [1977]), and in1986 two groups further characterized the existence of a humanserum-binding protein for GH (Baumann, G. et al., J. Clin. Endocrinol.Metab. 62:134-141 [1986]; Herington, A. C. et al., J. Clin. Invest.77:1817-1823 [1986]). When the GHBP was purified and characterized inthe rabbit its amino-terminal 37 residues were shown to be identical tothose of the extracellular part of the rabbit liver GH receptor (Spenceret al., J. Biol. Chem. 263:7862-7867 [1988]). It has been suggested thatthe GHBP derives from the membrane receptor by proteolytic cleavage(Trivedi and Daughaday, Endocrinology 123:2201 [1988]) or that it isproduced from a separate mRNA derived from the same gene as thefull-length GH receptor (Smith et al., Mol. Endo, p.984 [1989]; Baumbachet al., Genes and Dev. 3:1199 [1989]). Studies of the ontogeny of theGHBP activity in man (Daughaday et al., J. Clin. Endocrinol. Metab.65:1072-74 [1987]) and the changes in the concentrations of the GHBP andhepatic GH receptors in pregnant mice (Smith and Talamantes,Endocrinology 123:1489-94 [1988]) suggest that the serum GHBP levelscould be a peripheral indicator of GH receptor activity.

This idea that GHBP levels reflect GH receptor activity is supported bythe finding that patients with Laron-type dwarfism, a syndrome caused bythe lack of functional GH receptors, also lack GH binding activity inserum (Daughaday and Trivedi, Proc. Natl., Acad. Sci. USA 84:4636-40[1987]; Baumann et al., J. Clin. Endocrinol. Metabol 65:814-816 [1987];Laron et al., Acta Endocrinologica (Copenhagen) 121:603-608 [1989]).There are indications that in some patients with Laron type dwarfism theabnormality is caused by partial deletion of the GH receptor gene(Godowski et al., Proc. Natl. Acad. Sci. USA 86:8083-8087 [1989];Anselem et al., New England J. Med. 321:989-95 [1989]), which couldresult in growth failure due to inability to bind GH. For this type ofabnormality our assay would be particularly useful since it, in contrastto some immunoassays, would not detect the inactive protein. The serumGH binding capacity is reduced in Laron-dwarfism heterozygotes, and ithas been suggested that measurement of the hGHBP levels in serum couldbe of help for genetic counseling (Laron et al., supra).

Little is known about the physiological role of the GHBP, however,recent studies have shown that the binding protein can modify theeffects of growth hormone. It has been demonstrated that the GHBP altersthe distribution and half-life of GH (Baumann et al., Metabolism38:330-333 [1989]), and there is also evidence to suggest that the GHBPaffects the interaction of GH with its receptor on the target cells (Limet al., Endocrinology 1276:1287 [1990]). It has recently been shown thatrecombinant hGHBP produced in E. coli enhances the growth promotingeffects of hGH when given to GH deficient rats, indicating that the GHBPmay play an important role in the regulation of body growth in humans(see Example 5).

The LIFA of the present invention is used to monitor the concentrationof GHBP in the biological fluids of a patient to detect aberrantconcentrations. In Examples 5, 6 and 7 numerous applications of theassay are used to monitor and evaluate the activity of human GHBP aloneand complexed with hGH. These pharmacological applications includepurification, dosage, frequency of administration, and duration incirculation. The LIFA is also used to monitor the activity of hGHBP fromdifferent sources, such as E. coli, 293 cells or from natural tissuesources.

The LIFA has application in the pharmacologic evaluation of hGHBP actionin primates. The hGHBP with, and without, complexed hGH can be monitoredin primates to improve the dosage and frequency of administration.

In Primates

The ability of the GHBP to allow one to give infrequent GH injections,using similar GH doses to those used currently, yet maintain growthresponses, is of clinical significance. Experiments in the monkey, usingthe LIFA demonstrate that clearance of a GHBP+GH complex (in one of theforms described above or in a modified form), is delayed in primates.The clearance of injected GH bound to the GHBP is slowed to a degreesimilar to that which we have seen in the rat. This demonstrates inprimates the improved growth promoting activities of administering hGHcomplexed to the hGHBP.

In primates, including humans, the GHBP-complex is able to be given atinfrequent intervals, greater than every 2 days, more preferably atgreater than every 7 days, without a loss of efficacy compared toinjections of GH along every 1 or 2 days or daily for a week or more. Inaddition, the GHBP complexed with GH or alone will be given at lowertotal weekly doses compared to GH alone. The undesirable side effects ofGH treatment, for example the diabetogenic and fluid retainingproperties of GH, will be reduced with the use of the GHBP. There areother beneficial effects of using GH-GHBP complex including a heightenedIFG-1 response and the ability of the GHBP to direct GH preferentiallyto bone. This allows the GH-GHBP complex to be used for the treatment ofbone disorders, including the prevention and treatment of osteoporosis.In each situation the LIFA is used to monitor the progress of thereaction. The dosages, formulations and methods of using and makinghGHBP are described in U.S. Pat. 5,057,417.

The LIFA will be used to define groups of patients who have aberrantamounts of the GHBP complex. For example, a sub-set of poorly growingchildren, who are relatively resistant to the growth promoting activityof GH, will be found to be deficient in the GHBP. Such children includepatients with Turner's Syndrome, kidney disease, as well as a class ofbinding protein deficient patients who were previously described ashaving iodiopathic short stature. Pharmacokinetic studies delivering theGHBP or GHBP-GH complex subcutaneously, and assaying the blood levels ofGHBP-GH complex using the LIFA will be performed in man to establishsuitable temporal dosing regimes. Doses of GH-GHBP complex sufficient tostimulate rises in IGF-1 concentrations in blood will be determined andthese doses will indicate the doses of GHBP-GH complex necessary toinduce body growth. Subsequently, long-term studies in the GH-resistantchildren will be initiated to demonstrate the ability of the GHBP-GHcomplex to stimulate whole body growth, including bone growth. The LIFAwill be used to monitor blood levels of the GHBP.

A primary application of this invention is to use the GHBP LIFA tomonitor endogenous levels of GHBP before and during treatment for GH orGHBP deficiency. The assay of this invention serves to direct thetreatment that a patient undergoes. If there is no detectable GHBP inthe blood, a Laron-type syndrome may be present and IFG-1 treatmentindicated. If there is a low level of GHBP in the blood, additional GHor GHBP with or without IGF-1 may be indicated. Whatever treatmentregime (GH or GH+IFG-1, or GH-GHBP complex treatment) instituted theLIFA will be most valuable to determine the GHBP response to treatment.Blood GHBP concentrations will rapidly reflect the efficacy of GHtreatment much more so than measurement of traditional endpoints. A lackof response of blood GHBP levels will be used as a rapid diagnostic forconsidering alternative strategies for treatment.

Another use of the LIFA is to detect the biological activity ofendogenous blood GH. This assay uses the addition of a constant amountof GHBP followed by the addition of sample without saturating with GH.It is anticipated that patients will be detected who possess highimmuno-reactive but low immunofunctional concentrations of GH. A similarassay format can be used to measure the amount of bioactive GH in anysample, especially to test batches of recombinant GH for theirbiological activity.

The GHBP assay can be reversed to assay for GH. The captive antibodybinds GH, GHBP is complexed and the indicator antibody is specific forthe bound GHBP. This permits assay of GH alone or complexed with nativeGHBP.

Modes of Carrying Out the Invention

The present invention may be used to measure any known polypeptidehormone binding protein found in biological fluids. Similarly, it may beused with new hormone binding proteins as they are discovered. Among thepreferred binding protein targets of the assay method of the presentinvention are the following: any growth hormone binding protein,epidermal growth factor binding protein, insulin-like growth factorbinding proteins, insulin binding protein, corticotropin releasingfactor binding protein, nerve growth factor binding protein,transforming growth factor beta binding protein and activin bindingprotein. Detection of each of these binding proteins using the assaymethod of the present invention requires the use of their respectiveligand polypeptide hormone to saturate the hormone binding sites on thebinding protein. Polypeptide hormones may be purified from naturalsources, produced by solid phase protein synthesis or produced byrecombinant means. Among the preferred ligand polypeptide hormones usedare: growth hormone, epidermal growth factor, insulin-like growthfactor-1, insulin-like growth factor-2, nerve growth factor, insulin,corticotropin releasing hormone, transforming growth factor beta andactivin. The preferred binding proteins and polypeptide hormones may befrom any animal having such proteins in their biological fluids. Thebiological fluids may be any aqueous liquid such as the following:serum, plasma, lymph fluid, synovial fluid, follicular fluid, seminalfluid, amniotic fluid, milk, whole blood, urine, spinal fluid, saliva,sputum, tears, perspiration, mucus tissue culture medium, tissueextracts and cellular extracts.

The pH of the medium will usually be in the range of about 4-11, moreusually in the range of about 5-10, and preferably in the range of about6.4-9.5. The pH is chosen so as to maintain a significant level ofspecific binding by the HBP and the polypeptide hormone, the binding ofthe antibodies and the requirements of the detectable label. In someinstances, a compromise will be made among these three considerations.Various buffers may be used to achieve the desired pH and maintain thepH during the assay determination. Illustrative buffers include borate,phosphate, carbonate, Tris, barbital and the like. The particular bufferemployed is not critical to this invention, but in individual assays onebuffer may be preferred over another.

Moderate temperatures are normally employed for carrying out the assaymethod and usually constant temperatures are maintained during theperiod of the assay. The temperatures for the determination willgenerally range from about 4° to 50° C., more usually from about 15° to40° C.

The concentration of HBP which may be assayed will generally vary fromabout 10⁻⁴ -10⁻¹⁵, more usually from about 10⁻⁶ -10⁻⁻¹³ M. While theconcentrations of the various reagents will generally be determined bythe concentration range of interest of the HBP, the final concentrationof each of the reagents will normally be determined empirically tooptimize the sensitivity of the assay over the range of interest.

The LIFA may be used to monitor clinical administration of hGHBP, eithercomplexed with or without hGH. A preferred use of the assay method is ina method of promoting mammalian growth and anabolism comprising: (a)determining the optimal amount of a growth hormone binding proteinrequired to promote a growth hormone induced response; (b) measuring byLIFA the amount of growth hormone binding protein present in thebiological fluids of a person suspected of being deficient in saidinduced response; and, (c) comparing (a) with (b), and if (a) is greaterthan (b), administering growth hormone binding protein in an amountsufficient to increase the level of growth hormone binding protein tosaid optimal amount. Another preferred clinical application of the LIFAmethod is to monitor a growth hormone induced response such as weightgain, bone growth, muscle growth and function, organ growth andfunction, skin growth, and the level of IGF-1. The organs whose growthand function are stimulated may be thymus, kidney, liver, spleen, brainheart, lungs or gonads. Yet another application of the present LIFAmethod is for decreasing the frequency of injecting a growth promotingamount of a growth hormone binding protein-growth hormone complexcomprising: (a) determining the minimum necessary serum level of growthhormone-growth hormone binding protein complex required to maintainoptimal growth; (b) measuring by LIFA the level of growth hormonebinding protein present in a patient suspected of being deficient ingrowth hormone binding protein-growth hormone complex; and, if thecomplex level in (b) is less than the level in (a), then, (c)administering an amount of growth hormone being protein-growth hormonecomplex sufficient to maintain the level of complex for a period greaterthan two days. The period may be two to fourteen days, more preferablytwo to eight days, and most preferably three to seven days. The optimalamount of growth hormone binding protein is defined as that equal to orgreater than 90% of the average level found in healthy individuals.

Antibodies

While polyclonal antibodies may be used, the preferred antibody is amonoclonal antibody. Monoclonal antibodies are highly specific, beingdirected against a single antigenic site. The antibody used in thepresent invention must be specific for epitopes which do not interfereor block the binding of the polypeptide hormone with the hormone bindingprotein. Therefore, the capture or coat antibody must bind to epitopeson the binding protein which leave the hormone binding site availablefor hormone binding. Similarly, the detection antibody must be specificfor those polypeptide hormone epitopes which remain exposed followingbinding of the polypeptide hormone to the hormone binding protein. Theantibodies may be made by methods commonly available to those ofordinary skill in the art. Methods for the production of polyclonalantibodies are described in numerous immunology texts, such asMicrobiology, 3rd Edition, by Davis et al., Harper & Row, New York,1980.

Monoclonal antibodies may be produced by the method first described byKohler and Milstein, Eur. J. Immunol., 6:511 (1976). While the inventionis demonstrated using mouse monoclonal antibodies, the invention is notso limited; monoclonals from any animal species will also function inthe assay method of the present invention. In fact, in the present assaymethod, chimeric antibodies will also function. Chimeric antibodies aredescribed in U.S. Pat. No. 4,816,567, Morrison et al., Proc. Natl, AcadSci. USA, 81:6851 (1984); Neuberger et al., Nature 312:604 (1984);Takeda et al., Nature 314:452(1985). Chimeric antibodies are made bysplicing the genes from a mouse antibody molecule of appropriate antigenspecificity together with genes from another animal encoding a secondantibody. Similarly, monoclonal antibodies, or the antigen bindingregion of a monoclonal antibody, such as Fab or (Fab)₂ fragments, may beproduced by recombinant methods. Both chimeric and recombinantantibodies or antibody fragments may be used in the assay method of thepresent invention.

The monoclonal antibodies can belong to any of the classes or subclassesof antibodies, including IgA, IgD, IgE, IgG, and IgM. Actively bindingfragments of antibodies can also be employed, such as Fab, Fv, (Fab)₂,or the like. The monoclonal antibodies can be prepared by any convenientmeans which provides immortalization of the B-lymphocyte genesexpressing the antibody sub-units, such as fusion between sensitizedlymphocytes and a myeloid fusion partner; transformation, e.g., withEpstein-Barr virus (EBV); or other immortalization techniques.Alternatively, the genes can be isolated from a lymphocytic hostexpressing the antibodies and transferred to a more convenient host forexpression in accordance with known genetic engineering techniques.

The antibodies may be obtained from any convenient vertebrate source,such as murine, primate, lagomorpha, bovine, ovine, equine, canine,feline or porcine. The antibodies are often prepared by fusing spleencells from a host sensitized to the antigen and myeloma cell inaccordance with known techniques or by transforming the spleen cellswith an appropriate transforming vector to immortalize the cells. Thecells can be cultured in a selective medium, cloned, and screened toselect monoclonal antibodies that bind the designated antigens.

The methods used to produce the antibodies for the capture antibody andfor the detection antibody may be conveniently made by administering therespective immunogen, either binding protein or polypeptide hormone, andeliciting antibody. The preferred antibody is monoclonal antibody. Theantibody produced is then screened to determine the preferred antibodywhich does not hinder the binding reaction between the polypeptidehormone and the binding protein. The preferred monoclonal antibody fordetecting the hGH when bound to hGHBP was produced by a mouse hybridoma(Cunningham et al., Science (1989) 243:1330-1336). The anti-hGHBPmonoclonal antibody used as the capture or coat antibody is commerciallyavailable (Agen Inc., 90 East Halsey Road, Parsippanyu, N.J. 07054) orcan be made using the hGHBP as immunogen and screening the antibody asdiscussed above.

Because of the relative ease with which antibodies can now be preparedagainst antigens, preferred embodiments of the present invention usemonoclonal or polyclonal antibodies attached to the solid phase tocapture the HBP. Techniques for attaching specific HBP to various solidphase substrates, such as filter, plastic etc. are well known and neednot be described here in detail. See, for example, U.S. Pat. No.4,376,110 and the references cited therein. Among the more preferredcommon solid phase supports are: small sheets, plastic beads, assayplates or test tubes made from polyethylene, polystyrene, polypropyleneor other suitable material. Also useful are particulate materials suchas papers, agarose, cross-linked dextran, and other polysaccharides.

Interference by Heterophilic Antibodies

When results from GHBP assays using HRPO-conjugated MCB andHRPO-conjugated mouse polyclonal antibody were compared, one human serumsample showed much higher GHBP levels when detected with theMCB-conjugate. To test if the discrepancy could be due to heterophilicantibodies, purified mouse IgG (mlgG) at 0.5 mg/ml, was included in thesample buffer. The results showed that mlgG reduced the signal in sample#6 from 384 pmol/L to 215 pmol/L when assayed with the MAb-conjugate,while the GHBP concentrations were unchanged in two control samples (323vs 338, 92 vs 111 pmol/L when assayed with and without mlgGrespectively). These results indicate that the two different conjugatedantibodies were substantially the same if the unspecific binding wasblocked by the mlgG.

Sandwich type immunometric assays are subject to positive interferenceby heterophilic antibodies in the sample. This is caused by humananti-mouse antibodies in the human serum sample. Such heterophilicantibodies crosslink the coat and the conjugate mouse antibodies.Interference by heterophilic antibodies can be diminished or eliminatedif immunoglobulins from a nonimmunized animal (here to HBP or ligandhormone) are added to the assay to block the heterophilic antibodies inthe sample. In fact, the discrepancy in the GHBP concentration waseliminated when mouse IgG was included in the assay buffer. Thisindicated that the unspecific signal which was detected when theconjugated MAb was used as due to the present of anti-mouse IgGantibodies in the serum from this subject. Since one doesn't know whichsamples will show this type of interference it is best to always includemouse IgG in the first step of the assay.

Deposit of Hybridoma

The hybridoma cell line producing this anti-hGH antibody is HGH-B whichwas deposited with the American Type Culture (ATCC) 12301 ParklawnDrive, Rockville, Md., USA. on Nov. 9, 1990, and has ATTC Registrationnumber HB-10596. This deposit was made under the provisions of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedure and the Regulationsthereunder (Budapest Treaty). This assures maintenance of the viablecultures for 30 years from the date of the deposit. The cells will bemade available by the ATCC under the terms of the Budapest Treaty, andsubject to an agreement between Genentech, Inc. and the ATCC, whichassures permanent and unrestricted availability of the progeny of thecultures to the public upon issuance of the pertinent U.S. patent orupon laying open to the public of any U.S. or foreign patentapplication, whichever comes first, and assures availability of theprogeny to one determined by the U.S. Commissioner of Patents andTrademarks to be entitled thereto according to 35 USC 122 and theCommissioner's rules pursuant thereto (including 37 CFR 1.12 withparticular reference to 886 OG 638).

The assignee of the present application has agreed that if the cultureson deposit should die or be lost or destroyed when cultivated undersuitable conditions, they will be promptly replaced on notification witha viable specimen of the same culture. Availability of the depositedstrain is not to be construed as a license to practice the invention incontravention of the rights granted under the authority of anygovernment in accordance with its paten laws.

Detectable Markers

Detectable markers or labels on the antibodies that may be covalentlyattached include a suitable indicator such as an enzyme, radioactiveisotope, fluorescer, or other measurable tag as described in U.S. Pat.No. 4,275,149, bridging columns 19 to 28 and U.S. Pat. No. 4,318,980,columns 10-14. Of particular interest are enzymes which involve theproduction of hydrogen peroxide and the use of the hydrogen peroxide tooxidize a dye precursor to a dye. Particular combinations includesaccharide oxidases, e.g., glucose and galactose oxidase, orheterocyclic oxidases, such as uricase and xanthine oxidase, coupledwith an enzyme which employs the hydrogen peroxide to oxidize a dyeprecursor, that is, a peroxidase such as horse radish peroxidase,lactoperoxidase, or microperoxidase. Among the preferred enzymes are thefollowing: horseradish peroxidase, glucoamylase, alkaline phosphatase,glucose oxidase, and beta-D-galactosidase.

Additional enzyme combinations may be found in the subject matterdisclosed in the cited references. When a single enzyme is used as alabel, other enzymes may find use such as hydrolases, transferases, andoxidoreductases, preferably hyudrolases such as alkaline phosphatase andbeta-galactosidase. Alternatively luciferases may be used such asfirefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456).

Following the use of antibody specific for the ligand hormone, theamount of bound antibody is determined by measuring the activity of theattached indicator. In the case of enzymes, the amount of colordeveloped and measured will be a direct measurement of the amount ofhormone or hormone binding protein present. The conjugation of suchtags, including the enzymes, to an antibody as described herein is astandard manipulative procedure for one skilled in immunoassaytechniques. See for example, O'Sullivan et al., (1981) Methods for thePreparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay,in Methods in Enzymology (ed J. J. Langone & H. Van Vunakis), Academicpress, New York, Vol. 73, pp 147-166.

Kits

As a matter of convenience, the assay method of the present inventioncan be provide as a kit, i.e., a packaged combination with otherreagents for combination with a sample or samples in assaying for apolypeptide hormone binding protein and/or to determine relativesaturation with ligand polypeptide hormone. The components of the kitwill be provided in predetermined ratios. The kit will contain thespecific carrier solid phase for the capture antibody, capture antibodyseparate or bound to the carrier solid phase, the ligand hormone(preferably recombination), and the detection antibody containing adetectable label. Where the detectable label is an enzyme, the kit willinclude substrates and cofactors required by the enzyme, a dyeprecursor, which provides the detectable chromophore of fluorophore. Inaddition, other additives may be included such as stabilizers, buffersand the like. The relative amounts of the various reagents may be variedwidely to provide for concentrations in solution of the reagents whichsubstantially optimize the sensitivity of the assay. Particularly, thereagents may be provided as dry powders, usually lyophilized, includingexcipients, which on dissolution will provide for a reagent solutionhaving the appropriate concentration for combining with the sample to betested.

Definitions and Abbreviations:

Capture (coat) antibody: Antibody specific for hormone binding proteinepitope such that binding of antibody does not hinder the binding of theligand polypeptide hormone to the hormone binding protein. Antibody isattached to a solid phase and it is used to selectively bind to thebinding protein and facilitate removal of the binding protein from asolution.

Detection antibody: Antibody which is labeled with detectable markerspecific for an epitope on the polypeptide hormone and which is thedetectable marker.

HBP: hormone binding protein; the carrier protein found in biologicalfluids that has affinity for a ligand polypeptide hormone and acts as acarrier for the bound ligand polypeptide hormone.

hGH: human growth hormone, including multiple naturally occurring formssuch as 22 kd, 20 kd, placental variant (GH V), and variants produced byrecombinant methods (Cunningham et al., Science (1989) 243:1330-1336).

GHBP: growth hormone binding protein.

hGHBP: human growth hormone binding protein.

GH: growth hormone.

MAb: monoclonal antibody.

MCB: Monoclonal B produced by the mouse hybridoma HGH-B.

HRPO: horse radish peroxidase.

BSA: Bovine serum albumin.

LIFA: Ligand-mediated immunofunctional assay.

Purification of GHBP

The purification of the GHBP used in monitored using a conventionalELISA (Fuh, G. et al., J. Biol. Chem. 265, 3111-3115 [1990]). The LIFAof the present invention provides a better assay for detecting thepresent and assaying the amount of functional binding protein duringsuch a purification. The LIFA ensures that functional active bindingprotein rather than immunologically active binding protein is purified.During hGHBP storage the LIFA is used to follow the amount of functionalGHBP that remains in an active form with time. This property of theassay greatly aids in satisfying regulatory requirements concerning thestability of the GHBP during prolonged storage.

Materials and Methods

Selection of coat MAb

Four monoclonal antibodies to the rabbit liver growth hormone receptor,MAbs 1, 2, 5 and 7 were provided by Dr. M. Waters (University ofQueensland, Queensland, Australia), and an additional two MAbs (43 and263) were purified from mouse ascites fluid purchased from Agen,Australia. Immulon II removawell (Dynatech Laboratories, Inc.,Alexandria, Va.) were coated by incubating them overnight at 4° C. withantibody, 100 μL per well at 5 μg/mL in 50 mmol/liter carbonate buffer,pH 9.6 (coating buffer). After removing the coating solution,nonspecific binding sites on the coated plates were blocked with 150 μLof phosphate-buffered isotonic saline pH 7.2 (PBS) containing 5 g ofbovine serum albumin (BSA) per liter (blocking buffer), for 1 h at roomtemperature, followed by three washes with wash buffer (0.5 mL of Tween20 and 0.1 g of Thimerosal per liter of PBS). The immobilized MAbs wereevaluated in three different experiments. In the first experiment, usingsequential incubation steps, GHBP was first incubated with MAb coated onthe well, followed by addition of radiolabelled GH (GH-I¹²⁵). In thesecond experiment the reaction of GHBP and GH-I¹²⁵ was carried outsimultaneously in MAb coated wells. In the third experiment, GHBP waspre-incubated with hGH-I¹²⁵ overnight at 4° C. and then added to the MAbcoated well. In all three experiments, the nonspecific binding of tracerwas determined by substituting the GHBP solution in the reaction mixturewith incubation buffer (PBS containing per liter, 5 g of BSA, 0.5 mL ofTween 20 and 0.1 mL of Thimerosol).

Sequential Assay

In the sequential incubation experiment, 100 μL of GHBP (2.5 ng/100 μL)was allowed to react with immobilized MAb for 2 h, washed with washbuffer, and incubated for 4 h at room temperature with GH-I¹²⁵ (20,000cpm/100 μL). The wells were washed 6 times in wash buffer, blottedthoroughly on adsorbent paper and counted in a LKB series 1277 gammacounter (Pharmacia LKB Nuclear Inc., Gaithersburg, Md.) for 1 minute(FIG. 6).

Simultaneous Assay

In the simultaneous incubation experiment, 50 μL of GHBP at 2.5 ng/50 μLin incubation buffer and 50 μL of GH-I¹²⁵ at 20,000 cpm/50 μL inincubation buffer were incubated in each well for 4 h at roomtemperature. The wells were washed, blotted and counted as describedabove.

Preincubation Assay

In the pre-incubation experiment, 150 μL of GHBP at 2.5 ng/50 μL inincubation buffer and 150 μL of GH-I¹²⁵ at 20,000 cpm/50 μL inincubation buffer were incubated in test tube overnight at 4° C. Thereaction mixture was then added (100 μL per well) to the MAb coated welland incubated for 4 h at room temperature. The wells were washed,blotted and counted as previously described.

Enzyme-conjugated Anti-hGH Antibodies

The anti-hGH detection MAb was selected for conjugation because it didnot give detectable displacement of the GHBP and contains no overlappingdeterminants with the GHBP (Cunningham et al., Science 243:1330-1336[1989]). The antibodies were purified from ascites fluid using proteinA-Sepharose (Repligen Corp., Cambridge, Mass.) following establishedprocedures (Ey et al., Immunochem. 15:429 [1978]; Goding J. W., J.Immunol. Meth. 20:241 [1978]) and stored sterile in 0.01M sodiumphosphate, 0.15M sodium chloride, pH 7.2 (PBS) at 4° C. Purified MAbswere conjugated to horseradish peroxidase (Nakane and Kawaoi, J.Histochem. Cytochem. 22:1084 [1974]) and stored at -20° C. in 50%glycerol.

LIFA assay standards

The recombinant human growth hormone binding protein (GHBP) was purifiedfrom a mammalian cell line following the procedure outline by Spencer etal. (J. Biol. Chem. 263:7862-7867 [1988]). The purified GHBP amino acidcomposition as determined by quantitative amino acid analysis matchedwhat was theoretically expected for the cloned gene product. Thepurified GHBP was homogeneous based on analysis of SDS-gelelectrophoresis. The concentration of GHBP in the purified preparationwas established by Scatchard analysis (Scatchard, G., Annals of the NewYork Academy of Sciences 51:660-672 [1949]), and dilutions of thissample in PBS containing per liter, 5 g of BSA, 5.0 mM EDTA, 0.5 ml ofTween 20 and 0.1 g of Thimerosal (assay buffer) were then used asstandards in the LIFA. GHBP produced in E. coli was also used, butsurprisingly found to give non-parallel dilution curves in the assay.Therefore, the preferred GHBP is either natural GHBP or GHBP produce bycells which produce GHBP in a native configuration, that is,glycosylated.

Recombinant human GH

Recombinant human growth hormone (GH) was supplied by Genentech Inc.,South San Francisco, Calif., USA.

Serum and plasma samples

Serum and plasma (EDTA, citrate and heparin as anticoagulants) sampleswere obtained from healthy adult volunteers (9 men and 7 women, 26 to 43years old). The samples were centrifuged and stored at -70° C. untilassayed.

Therapeutic Treatment Following GHBP Evaluation

For the various purposes of this invention, the COMPOSITIONS (GHBP+GH,GH, IGF-I, IGF-1+binding protein) administered to the mammal or avian byany suitable technique, including parenterally, and can be administeredlocally or systemically.

The COMPOSITIONS are directly administered to the mammal by any suitabletechnique, including parenterally, intranasally, or orally. The specificroute of administration will depend, e.g., on the medical history of thepatient, including any perceived or anticipated side or reduced anaboliceffects using hGH or IGF-I alone, and the growth defect to be corrected.Examples of parenteral administration include subcutaneous,intramuscular, intravenous, intraarterial, and intraperitonealadministration. Most preferably, the administration is by continuousinfusion (using, e.g., minipumps such as osmotic pumps), or be injectionusing, e.g., intravenous or subcutaneous means. Preferably, theCOMPOSITIONS administration is subcutaneous. The administration may alsobe as a single bolus or by slow-release depot formulation. Mostpreferably, the IGF-I or IGF-1 plus binding protein is administeredcontinuously by infusion, most preferably subcutaneously; GHBP+GH or GHalone is administered daily subcutaneously by injection. Mostpreferably, the GHBP+GH is administered intermittently every 2 or moredays, weekly, biweekly, or monthly.

The IGF-I is suitably administered together with its binding protein,for example, BP53, which is described in WO 89/09268 published Oct. 5,1989 and by Martin and Baxter, J. Biol. Chem., 261: 8754-8760 (1986),the disclosures of which are incorporated herein by reference. Thisprotein is an acid-stable component of about 53 Kd on a non-reducingSDS-PAGE gel of a 125-150 Kd glycoprotein complex found in human plasmathat carries most of the endogenous IGFs and is also regulated by GH.The IGF-I is also suitably coupled to a receptor or antibody or antibodyfragment for administration. Similarly, the GH can be delivered coupledto another agent such as an antibody, an antibody fragment, or one ofits binding proteins.

The COMPOSITIONS to be used in the therapy will be formulated and dosedin a fashion consistent with good medical practice, taking into accountthe clinical condition of the individual patient (especially the sideeffects of treatment with hGH of IGF-I along or growth retardation aftercontinuous GH treatment), the site of delivery of the COMPOSITIONS, themethod of administration, the scheduling of administration, and otherfactors known to practitioners. The "effective amounts" of eachcomponent for purposes herein are thus determined by such considerationsand must be amounts that enhance the anbolic growth of the treatedpatient.

As a general proposition, the total pharmaceutically effective amount ofeach of the COMPOSITIONS administered parenterally per dose will be inthe range of about 1 μg/kg/day to 50 mg/kg/day of patient body weight,although, as noted above, this will be subject to a great deal oftherapeutic discretion. More preferably, this dose is at least 2μg/kg/day, and most preferably at least 5 μg/kg/day for each hormone. Ifgiven continuously, the IGF-I, IGF-1+binding protein, GHBP+GH and GH areeach typically administered at a dose rate of about 1 μg/kg/hour toabout 100 μg/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a minipump. An intravenousbag solution may also be employed. The key factor in selecting anappropriate dose is the anabolic result obtained, as measured byincreases in body weight gain, lean body mass, or statutory growthapproximating the normal range, or by other criteria for measuringanabolic activity as defined herein as are deemed appropriate by thepractitioner.

The COMPOSITIONS are also suitably administered by sustained-releasesystems. Suitable examples of sustained-release compositions includesemi-permeable polymer matrices in the form of shaped articles, e.g.,films, or microcapsules. Sustained-release matrices include polylactides(U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (U. Sidman et al., Biopolymers, 22, 547-556(1983)), poly(2-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed.Mater. Res., 15: 167-277 (1981), and R. Langer, Chem. Tech., 12: 98-105(1982)), ethylene vinyl acetate (R. Langer et al., Id.) orpoly-D--(-)--3--hydroxybutyric acid (EP 133,988). Sustained-releasecompositions also include liposomally entrapped COMPOSITIONS. Liposomescontaining COMPOSITIONS are prepared by methods known per se: DE3,218,121; Epstein et al., Proc. Natl. Acad. Sci. U.S.A., 82: 3688-3692(1985); Hwang et al., Proc. Natl. Acad. Sci. U.S.A., 77: 4030-4034(1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641;Japanese Pat. Appln. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545;and EP 102,324. Ordinarily, the liposomes are of the small (about200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. percent cholesterol, the selected proportionbeing adjusted for the optimal COMPOSITIONS therapy.

For parenteral administration, in one embodiment, the IGF-I and GH areformulated generally by mixing each at the desired degree of purity, ina unit dosage injectable form (solution, suspension, or emulsion), witha pharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to polypeptides.

Generally, the formulations are prepared by contacting the COMPOSITIONSeach uniformly and intimately with liquid carriers or finely dividedsolid carriers or both. Then, if necessary, the product is shaped intothe desired formulation. Preferably the carrier is a parenteral carrier,more preferably a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, and dextrose solution. Non-aqueous vehicles such asfixed oils and ethyl oleate are also useful herein, as well asliposomes.

The carrier suitably contains minor amount of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine,; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium, and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

The COMPOSITIONS are each typically formulated individually in suchvehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably1-10 mg/ml, at a pH of about 4.5 to 8. Full-length IGF-I is generallystable at a pH of no more than about 6; des(1-3)- IGF-I is stable atabout 3.2 to 5; hGH and GHBP are stable at a higher pH of, e.g.,6.0-7.8. It will be understood that use of certain of the foregoingexcipients, carriers, or stabilizers will result in the formation ofIGF-I or GH salts.

In addition, the COMPOSITIONS preferably the full-length IGF-I, aresuitably formulated together in a suitable carrier vehicle to form apharmaceutical composition that does not contain cells. In oneembodiment, the buffer used for formulation will depend on whether thecomposition will be employed immediately upon mixing or stored for lateruse. If employed immediately after mixing, the COMPOSITIONS can beformulated in mannitol, glycine, and phosphate, pH 7.4. If this mixtureis to be stored, it is formulated in a buffer at a pH of about 6, suchas citrate, with a surfactant that increases the solubility of the GH atthis pH, such as 0.1% polysorbate 20 or poloxamer 188. The finalpreparation may be a stable liquid or lyophilized solid.

The COMPOSITIONS to be used for therapeutic administration must besterile Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). TherapeuticCOMPOSITIONS (IGF-I, IGF-1+binding protein, GHBP+GH compositions)generally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

COMPOSITIONS ordinarily will be stored in unit or multi-dose containers,for example, sealed ampoules or vials, as an aqueous solution or as alyophilized formulation for reconstitution. As an example of alyophilized formulation, 10-ml vials are filled with 5 ml ofsterile-filtered 1% (w/v) aqueous GH solution, and the resulting mixtureis lyophilized. The infusion solution is prepared by reconstituting thelyophilized GH using bacteriostatic Water-for-Injection.

GHBP plus GH ordinarily will be stored in unit or multi-dose containers,for example, sealed ampoules or vials, as an aqueous solution or as alyophilized formulation for reconstitution. As an example of alyophilized formulation, 10-ml vials are filled with 5 ml ofsterile-filtered 1% (w/v) aqueous IGF-I solution, and the resultingmixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized GHBP plus GH using bacteriostaticWater-for-Injection. The GHBP+GH formulation may further containmannitol, glycine, a buffer, and a non-ionic surfactant. The formulationof the subject invention may optionally include one of several types ofnon-ionic surfactants, such as the polysorbates (e.g. polysorbate 20,80, etc.) and the poloxamers (e.g. poloxamer 188). When polysorbate 80is used the molar ratio of GHBP+GH:polysorbate 80 is 1:0.07-30,advantageously 1:0.1-10, and most advantageously 1:3. On a weight tovolume basis, polysorbate 80 is added in amounts of about 0.001 to about2% (w/v), in order to enhance further the stability of the hGH.Polysorbate 80, in concentrations above 0.01% (w/v) reduces the amountof aggregation forming upon lyophilization. In addition to improvedshell life, the surfactant containing formulation of the subjectinvention inhibits the formation of protein aggregates when thereconstituted formulation is shaken.

When GHBP+GH is administered, it must contain one or more of its bindingproteins. A well characterized such binding protein is the high-affinitygrowth hormone binding protein (GHBP) constituting the extracellulardomain of the GH receptor that circulates in blood and functions as aGHBP in several species [Ymer and Herington, Mol. Cell. Endocrino.,41:153 (1985); Smith and Talamantes, Endocrinology, 123: 1489-1494(1988); Emtner and Roos, Acta Endocrinologica (Copenh.), 122: 296-302(1990)], including man (Baumann et al., J. Clin. Endocrinol. Metab., 62:134-141 (1986); EP 366,710 published 9 May 1990; Herington et al., J.Clin. Invest., 77: 1817-1823 (1986); Leung et al., Nature, 330: 537-543(1987)). A second BP with lower affinity for GH has also been describedthat appears to be structurally unrelated to the GH receptor (Baumannand Shaw, J. Clin. Endocrinol. Metab., 70: 680-686 (1990)).

Novel formulations of zinc, GHBP and GH result in a stable compositionsuitable for prolongs storage, and for therapeutic administration.Therapeutic formulations containing the Zn²⁺ ion are stable allowingtherapeutic administration of the formulation. The formulation aspect ofthe present invention is thus directed to such formulations, and to allassociated formulations and as a means for effectively stabilizingGHBP+GH. The formulation contains zinc, and substantially pure GHBP andGH free of contaminating proteins or infectious agents found in humans.Formulations of the present invention may additionally contain apharmaceutically acceptable buffer, amino acid, bulking agent and/ornon-ionic surfactant. These include, for example, buffers, chelatingagents, antioxidants, preservatives, cosolvents, and the like; specificexamples of these could include, trimethylamaine salts ("Tris buffer"),and disodium edetate.

hGH Compositions

As used herein, the terms "human growth hormone" or "hGH" denote humangrowth hormone produced, for example, from natural source extraction andpurification, and by recombinant cell culture systems. The nativesequence of hGH and its characteristics are set forth, for example, inHormone Drugs, Gueriguigan et at., U.S.P. Convention, Rockville, Md.(1982) The terms likewise cover biologically active human growth hormoneequivalents; e.g., differing in one or more amino acid(s) in the overallsequence. Further, the terms as used in this application are intended tocover substitution, deletion and insertion amino acid variants of hGH,or post translational modifications Examples of such variants aredescribed in PCT Pub. WO90/04788 published 3 May 1990. The hGH used inthe formulations of the present invention is generally produced byrecombinant means as previously discussed. The formulation ofrecombinant GHBP+GH is substantially pure, free of other human proteins,free of infectious agents such as the human immunodeficiency virus (HIV)and it is soluble. "Substantially pure" GHBP+GH means GHBP and GH thatis free of proteins with which it ordinarily is associated in bodilyfluids such as blood, plasma and serum. Ordinarily, substantially puremeans GHBP and GH which is greater than about 95% pure by weight oftotal protein, and preferably greater than 98% pure by weight.

Formulation Amino acids.

In an alternative formulation embodiment, a pharmaceutically acceptableamino acid, for example glycine, is added to the GHBP and GH:zinc ionformulation. When glycine is present, the molar ratio of GHBP+GH:glycineis 1:5-600 In addition to glycine, amino acids such as alanine,glutamine, asparagine, arginine or lysine or derivatives of such aminoacids may be used in the subject formulation. Such amino acids areparticularly advantageous when lyophilizing the formulation to create asufficient mass to form a stable, dry caked formulation.

Non-Ionic Surfactant

In another embodiment a non-ionic surfactant is added to the GHBP+GHformulation. The formulation of the subject invention may optionallyinclude one of several types of non-ionic surfactants, such as thepolysorbates (e.g. polysorbate 20, 80 etc.) and the poloxamers (e.g.poloxamer 188). Advantageously polysorbate 80 is used, and the molarratio of hGH: polysorbate 80 may be 1:0.03-60. On a weight to volumebasis, polysorbate 80 is added in amounts of about 0.001 to about 2%(w/v), in order to enhance further the stability of the GHBP and GH.Polysorbate 80, in concentrations above 0.01% (w/v) may reduce theamount of inactive aggregates forming upon lyophilization andreconstitution. The use of non-ionic surfactants improves formulationstability when exposed to shear and surface stresses without causingdenaturing of the protein. Further, such surfactant containing GHBP andGH formulations, may be employed in aerosol devices such as those usedin a pulmonary dosing, and needleless jet injector guns. Such deliveryformulations may be improved by the addition of non-ionic surfactants inthe range of 0.1-5% (w/v).

As used herein, the expression mammal refers to any mammal butespecially primates, bovine, ovine, canine, feline, equine and rodentia.Specifically it includes human, cows, horses, rats, mice, rabbits,monkeys, cats, dogs, pigs. The term avain refers to any bird,particularly chicken, turkey, duck and goose.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the constructs deposited,since the deposited embodiment is intended to illustrate only certainaspects of the invention and any constructs that are functionallyequivalent are within the scope of this invention. The deposit ofmaterial herein does not constitute an admission that the writtendescription herein contained is inadequate to enable the practice of anyaspect of the invention, including the best made thereof, nor is it tobe construed as limiting the scope of the claims to the specificillustrations that they represent. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims. The following examples areintended to illustrate the best mode now known for practicing theinvention, but the invention is not to be considered limited to theseexamples.

EXAMPLE 1 Selection of GHBP Assay Antibody

Two distinct antibodies are required for the LIFA method. The firstantibody is a capture antibody which coats the solid phase and is usedto selectively remove the HBP from the biological sample being assayed.This antibody must be specific for epitopes which do not hinder thebinding of the ligand hormone. The second detection antibody is specificfor an epitope on the ligand hormone. This second detection antibodymust bind the ligand hormone at a site that does not hinder its abilityto complex with the HBP. In the case of GHBP, known commerciallyavailable monoclonal antibody were screened for the desired bindingproperties. The detection monoclonal antibody specific for hGH was newlycreated in a mouse hybridoma system.

Coat MAb selection

For assaying for the present of GHBP a capture antibody which binds GHBPis needed to coat the solid phase, in this case a microtiter plate well,for binding the GHBP. Five mouse anti-GHBP MAbs (1, 5, 7, 43, and 263)were evaluated at their optimal coat concentration, first to determinetheir binding sites on GHBP relative to GHBP-GH i.e. sequential assayand their capacity to bind to GHBP in the presence of circulatingGH-I¹²⁵ (simultaneous incubation format). They were then examined basedon their capacity to bind to GHBP-GH-I¹²⁵ complex (pre-incubationexperiment). Since both MAbs 1 and 7 show very weak binding in thesequential and simultaneous assay format, only MAbs 5, 43 and 263 weretested in the pre-incubation experiment. FIG. 2 shows the percent boundfor each MAb under three different assay configurations. The data showthat MAb 263, which gave the highest bound in all three conditions isthe most suitable MAb as coat since it is able to bind to free GHBP aswell as GHBP in complex with GH. More importantly, the sequentialincubation experiment showed that the MAb 263 did not interfere with thepresent hGHBP-hGH binding site.

FIG. 3 shows a comparison of two standard curves generated with andwithout preincubation with hGH. One set of standards were incubated withhGH (final concentration 200 ng/ml) over night at 4° C., the controlstandards were incubated with assay buffer. The samples so generatedwere then assayed in the LIFA according to the standard protocol i.e.all samples were exposed to hGH on the microtiter plates. As seen inFIG. 3, similar results are obtained whether or not the GHBP ispreincubated with hGH. The binding of the GHBP to the coat antibody andthe saturation of the GHBP with it's ligand can be carried oursimultaneously by coincubating the samples and the hGH in the microtiterwells. The simplified LIFA gave a standard curve similar to thatobtained with two separate steps but required only half the incubationtime (2 h vs 4 h, FIG. 1, third box down).

The MCB, see below (Detection MAb Selection), which was used forHRPO-conjugation, binds to the 22 kDa--GH with high affinity, but hasvery low affinity for the 20 kDA and the possible interference by 20kDA--GH in the assay was consequently tested. FIG. 4 shows that theaddition of 200 ng/ml of the 20 kDa--hGH to the 200 ng/ml hGH solutionresults in a standard curve similar to that obtained by incubation with200 ng/ml of hGH. This shows that the 20 kDa--GH does not interfere inthe present GHBP assay to any substantial degree.

Detection MAb Selection

For assaying for the present of hGH, a detection antibody is needed withhigh affinity for an epitope on the hGH which will not hinder thebinding of the hGH to the GHBP. Monoclonal antibody was made in a mousesystem using recombinant hGH and screened for the required specificity.The best mouse monoclonal antibody was produced by a hybridomadesignated HGH-B. This hybridoma was deposited with the ATCC aspreviously discussed.

EXAMPLE 2 LIFA Assay Procedure for GHBP

The LIFA assay procedure developed for measuring the GHBP is as follows.Ninety-six-well microtiter plates (Corning Glass Works, Corning, N.Y.)were coated with MAb 263 by incubating overnight at 4° C. with 100μl/well of antibody at 20 μg/ml in 50 mmol/liter of sodium carbonatebuffer, pH 9.6 (coat buffer) (see FIG. 1, step 1). After removal of thecoating solution, the coated plates were blocked with 150 μl per well of5 g/liter of BSA in PBS for 1 h at room temperature (FIG. 1, step 2),and washed six times with 0.5 g/liter of Tween 20 in PBS (wash buffer).

Standards diluted in assay buffer or samples (50 μM serum or plasma and50 μM assay buffer) were dispensed onto the coated wells (100 μl/well)(FIG. 1, step 3). Plates were sealed and incubated at room temperaturefor 2 h with gentle agitation. Plates were washed six times with washbuffer. Recombinant hGH at 200 ng/ml or assay buffer was then added (100μl/well) FIG. 1, step and incubated at room temperature for 2 h. Plateswere washed six times with wash buffer before additiojn of horseradishperoxidase (HRPO) labelled MAb MCB (100 μl/well) 4). After furtherincubation for 2 h at room temperature, the plates were washed six timeswith wash buffer. Freshly prepared substrate solution (0.4 g ofo-phenylendiamine dihydrochloride in one liter of PBS plus 0.4 ml of 30%hydrogen peroxide) was added to the plates (100 μl per well) andincubation carried out in the dark for 15 min at room temperature FIG.1, step 5. The reaction was stopped by the addition of 100 μl of 2.25mol/L sulfuric acid and absorbance at 490 nm determined on a Vmax platereader (Molecular Devices, Menlo Park, Calif.). A standard curve wasgenerated by plotting absorbance vs. log of GHBP concentration, using a4-parameter nonlinear regression curve fitting program. Sampleconcentrations were obtained by interpolation of their absorbance on thestandard curve.

EXAMPLE 3 Properties of the GHBP Assay

The GHBP assay was evaluated and the range, sensitivity, specificity,and precision of the assay determined.

The specificity of this assay was tested by substituting the GHBP withfour other soluble receptors (rCD4, rHER2 ECD, EGF-receptor andrPRL-receptor) and an unrelated protein produced in CHO cells (HIVenvelope protein, gp120). All four proteins were obtained from GenentechInc. The results (Table 1) showed that the assay has less than 0.01%cross-reactivity with these proteins. In addition, cross-reactivity withhuman placental lactogen (HPL) and human prolactin (PRL) was tested.Substituting HPL and PRL for hGH at the same concentration (200 ng/ml)resulted in values undistinguishable from the blanks when when testedtogether with the GHBP or PRL-receptor.

                  TABLE 1                                                         ______________________________________                                        CROSS-REACTIVITY                                                                       Concentration                                                                   Tested   Measured    Cross-reactivity.sup.a                        Protein Tested                                                                           (μg/ml)                                                                             (ng/ml).sup.b                                                                             %                                             ______________________________________                                        rHER2 ECD  10.0     <0.6        <0.01                                         rCD4       10.0     <0.6        <0.01                                         rgp120     10.0     <0.6        <0.01                                         EGF-receptor                                                                             10.0     <0.6        <0.01                                         rPRL-receptor                                                                            10.0     <0.6        <0.01                                         ______________________________________                                         .sup.a Percent crossreactivity was calculated as concentration                measured/amount tested × 100.                                           .sup.b The concentration is claculated by using 26 kD as the molecular        weight of the rhGHBP                                                     

Assay precision

Serum samples with low, medium, or high GHBP concentrations wereanalyzed in 24 replicates for the assessment of intra-assay precision(Table IIA). Interassay precision was determined by measuring samples oflow, medium or high GHBP concentrations in ten separate experiments(Table 2B). The coefficients of intra-assay variation at all threelevels ranged from 6.3 to 8.9% while the coefficients of interassayvariation ranged from 9.7 to 12.9%.

                  TABLE 2                                                         ______________________________________                                        PRECISION OF THE LIFA                                                                     Sample 1 Sample 2 Sample 3                                        ______________________________________                                        A. Intra-assay Precision                                                      Replicates    24         24       24                                          Mean (pmol/L) 138        268      614                                         S.D. (pmol/L) 9.2        16.9     54.6                                        C.V. (%)      6.6        6.3      8.9                                         B. Interassay Precision                                                       Replicates    10         10       10                                          Mean (pmol/L) 136        293      674                                         S.D. (pmol/L) 17.6       33.1     65.0                                        C.V. (%)      12.9       11.3     9.6                                         ______________________________________                                    

Linearity of the assay

The linearity of the assay was determined by making serial dilutions ofserum samples in assay buffer and measuring the concentration of GHBP.Results were examined by correlating the observed concentrationdetermined in the LIFA with the calculated concentration obtained bymultiplying the dilution factor with the concentration of the undilutedsample determined in the LIFA. Linear regression analysis of the samplesresulted in correlation coefficients of 0.99 or greater (FIG. 5),indicating that the assay is linear.

EXAMPLE 4 Determining GHBP in Biological Fluids

Spike recovery

Purified GHBP at three different concentrations in assay buffer wereadded in equal volume to four serum samples and four plasma samples. Thegenerated samples were assayed in the LIFA. The theoreticalconcentration was calculated for each mixed sample and was used tocalculate percent recovery. The data in Table 3A and Table 3B show anaverage recovery of 106.7 and 99.5 for serum and plasma respectively,with a range of 89.1 to 115.9%, demonstrating the accuracy of the assay.

Saturation of the GH-BP with GH

FIG. 3 shows a comparison of two standard curves generated with andwithout preincubation with GH. One set of standards were incubated withGH (final concentration 200 ng/ml) overnight at 4° C., the controlstandards were incubated with assay buffer. The samples so generatedwere then assayed in the LIFA according to the standard protocol, i.e.all samples were exposed to GH on the microtiter plates. As seen in FIG.2, similar results are obtained whether or not the GHBP is preincubatedwith GH.

Application of the LIFA method

Total and GH-bound GHBP levels in random serum samples from 16 healthyadults and two patients with Laron type dwarfism are shown in FIG. 6.GH-BP levels were detectable in samples from all normal subjects (FIG.6; patients 1-16). In contrast, GHBP concentrations were undetectable(<30 pmol/L) in both patients with Laron-type dwarfism (FIG. 6: patients#17, #18).

                  TABLE 3A                                                        ______________________________________                                        ACCURACY.sup.a in serum                                                       Spiked  Serum                                                                 Sample.sup.b                                                                          Sample.sup.b                                                                            Expected.sup.c                                                                          Observed                                          (pmole/L)                                                                             (pmole/L) (pmole/L) (pmole/L)                                                                             % Recovery                                ______________________________________                                        204     177       192       212     110.4                                             238       219       254     115.9                                             465       225       362     105.9                                             1138      671       735     109.5                                     658     177       419       412     97.6                                              238       446       500     111.1                                             465       562       577     101.8                                             1138      898       962     107.1                                     1427    177       802       762     95.0                                              238       832       931     112.0                                             465       946       965     102.1                                             1138      1282      1438    111.9                                                                 Average 106.7                                                                 Range   95.0-115.9                                ______________________________________                                         .sup.a Equal volumn of three purified rGHBP (column #1) were each added t     serum samples (column #2) and assayed in the LIFA.                            .sup.b GHBP concentrations had been determined previously by LIFA.            .sup.c (spiked + serum)/2                                                

                  TABLE 3B                                                        ______________________________________                                        ACCURACY.sup.a in plasma                                                      Spiked  Plasma                                                                Sample.sup.b                                                                          Sample.sup.b                                                                            Expected.sup.c                                                                          Observed                                          (pmole/L)                                                                             (pmole/L) (pmole/L) (pmole/L)                                                                             % Recovery                                ______________________________________                                        142     100       121       112     92.6                                              150       146       154     105.5                                             300       221       231     104.5                                             569       355       335     94.4                                      435     100       267       254     95.1                                              150       292       323     110.6                                             300       367       327     89.1                                              569       502       500     99.6                                      835     100       467       458     98.1                                              150       492       538     109.3                                             300       567       531     93.6                                              569       702       715     101.9                                                                 Average 99.5                                                                  Range   89.1-110.6                                ______________________________________                                         .sup.a Equal volumn of thred purified rGHBP (column #1) were each added t     plasma samples (column #2) and assayed in the LIFA.                           .sup.b GHBP concentrations had been determined previously by LIFA.            .sup.c (spiked + plasma)/2                                               

EXAMPLE 5 Monitoring of GHBP in Growth Promotion

The LIFA of the present invention may be used to monitor theconcentration of GHBP in the biological fluids of a patient, and if thelevel is inadequate for the desired rate of growth, additional GHBPadministered. An example of low GHBP levels is in Laron dwarfism;whereas a high GHBP levels may be present in patients with excess GHsecretion. If the level GHBP is insufficient for the desired rate ofgrowth, additional GHBP alone or complexed to hGH may be administered.The optimal level of GHBP may be determined by the methods discussedabove, in combination with measuring the level of GHBP in a series ofnormal healthy individuals. GHBP may be evaluated in any mammaliansystem, preferably in rodents and primates.

In Rodents

Two rodent models of GH deficiency are used: 1) rats where the glandproducing GH, the pituitary, is surgically removed (hypophy-sectomizedrats) and 2) animals genetically deficient in growth hormone (dwarfrats, Charlton, H. W. et al., J. Endo. 119:51-58 [1988]). These rats aretreated with human GH (hGH) along or hGH coupled to human GHBP which isproduced recombinantly in E. coli or alternatively in mammalian 293cells. Several indices of growth. Monitoring of the level of GHBP and GHis required to determine the metabolic fate of administered GHBP andcomplexed GH.

Hypophysectomized Rats

Recombinant hGHBP and hGH are given either alone or in combination tohypophysectomized rats, a recognized model for measuring GH bioactivity(Thorngren, K. B. & Hansson L. I. Acta. Endo. 75:653-668 [1977]). HumanGH (0.03, 0.1 and 0.3 mg/kg, as 7 daily injections) induces adose-related weight gain while injections of E. coli--derived hGHBP atthese same 3 doses produces no effect by itself. However,co-administration of 0.3 mg/kg hGHBP with 0.1 mg/kg hGH not only givesgreater weight gain than 0.1 mg/kg hGH along (p<0.01), but also inducesgreater weight gain than three times more hGH (22.0±3.6 vs 17.1±2.1 grespectively; mean±s.d., p<0.01). Longitudinal bone growth parallelsbody weight gain. Thus, co-administration of 0.3 mg/kg hGHBP and 0.1mg/kg hGH gives greater bone growth than 0.3 mg/kg hGH (102±14 vs 84±17microns/day; p<0.05), and 0.1 mg/kg hGHBP plus hGH gives greater bonegrowth than hGH alone (99±6 vs 72±17 microns/day; p<0.01).

The liver, spleen and kidney are all significantly larger followingco-administration of hGHBP (0.3 mg/kg) with hGH (0.1 mg/kg) than with0.1 mg/kg hGH p<0.05), and kidney (836±60 vs 716±57 mg; p<0.05). Theweights of liver, spleen and kidney in excipient treated rats are4.5±0.2 g, 193±32 mg, and 687±58 mg, respectively. These responses tohGH are at least doubled by hGHBP. The serum concentrations of IGF-1 andhGH 24 h after the last injection are markedly elevated byco-administration of the two highest doses of hGHBP with hGH, whilehGHBP causes as much as 20-fold more hGH to be present after 24 h.

An ELISA (Fuh, G. et al., J. Biol. Chem. 265:3111-3115 [1990]) for hGHBPadapted for use in serum shows the reason for the persistence of the GHin the blood 24 hours after the seventh subcutaneous bolus injection wasgiven to the rats. The hGHBP is only detectable in the animalsco-administered hGHBP and hGH. When the GHBP is given alone itdisappears from the blood more rapidly than when the GHBP is givencomplexed to GH. These findings from measuring the GHBP in blood suggestthat it is the persistence of the GH+GHBP complex in the blood of therats that causes many or all of the above improved activities of GH. TheLIFA method of the present invention is therefore used to follow GHBPduring its preparation, storage, use and in body fluids following GHBPadministration.

Dwarf Rat System

We also compared hGH and hGHBP in a dwarf rat which has a pituitary GHcontent 5-10% of normal, grows slowly, and responds to GH treatment(Charlton, H. W. et al., J. Endo. 119: 51-58 [1988]) Co-administrationof 0.27 mg/kg of hGHBP with 0.27 mg/kg hGH increases weight gaincompared to 0.27 mg/kg hGH alone (11.1±4.2 vs 7.5±1.7 g; p<0.05).Co-administration of all three doses of hGHBP with 0.27 mg/kg of hGHsignificantly increasing bone growth compared to 0.27 mg/kg hGH along(low 33.5±5.8, medium 38.6±8.6, high 35.5±5.0, vs 26.0±4.1 microns/day;p<0.05). Serum IGF-1 concentrations are elevated by co-administrationeven compared to 0.81 mg/kg hGH alone (high hGHBP 136±45 vs 90±16 ng perml; p<0.05) as were hGH concentrations (high hGHBP 609±240 vs 73±22 pgper ml; p<0.0001).

GHBP from Mammalian 293 Cells

In marked contrast, hGHBP produced in human 293 cells completelyinhibits GH responses in hypophysectomized rats. Weight gains after 10daily s.c. injections of hGH along are 11.3±2.5, 16.4±2.1 and 21.1±2.1 gat 0.03, 0.1, and 0.3 mg/kg/day respectively. When hGH at 0.03 and 0.1mg/kg/day is co-administered with a 2-fold molar excess of 293derivedhGHBP this weight gain is abolished (3.0±2.1 and 3.0±1.6 g,respectively) compared to the hGH and excipient (2.3±1.6 g) groups. Thisdifference between the growth responses induced using these 2 forms ofhGHBP may be due to a difference in hGH clearance from the circulationwhich is reduced about 10 fold for GHBP derived from E. coli (Moore, J.A. et al., Proc. US Enocr. Soc. 71, Abstract #1652 [1989]) or purifiedform natural sources (Baumann, G. & Shaw, M. A., J. Clin Endocrinol.Metab., 70:680-686 [1990]; Baumann, G., Shaw, M. A. & Buchanan, T. A.,Metabolism. 38:338-333[1989].

The clearance (ml/min/kg) of hGH in normal male rats, following an i.v.bolus of hGH alone is 18.6±3.4, for hGH co-administered with GHBP fromrabbit sera is 2.1±0.2, for GHBP from E. coli 1.9±0.4, or from 293 cells41.3±16.7. Therefore, for the 293-derived hGHBP the clearance of hGH isincreased two fold, suggesting a correlation between in vivo potency andhGH clearance. The decreased clearance of hGH complexed to the E.coli--derived hGHBP may be due to the complex being of sufficient size(Mr>40 kDa) to escape filtration by the kidney. Proteins produced in 293cells can have heterogenous carbohydrate patterns, possibly due toincomplete glycosylation, causing them to be rapidly cleared by theliver, which may explain the rapid clearance of 293-derived hGHBP.

In this instance the 293-derived GHBP therefore acts as an inhibitor ofGH action, compared to the enhancing activities of the E. coli-derivedprotein. This difference in activity appears to be due to the differingclearances of the two molecules from the blood. The present inventionaids in similarly discriminating inhibitory and stimulatory bindingproteins on the basis of their clearance from the blood.

Persistence of GHBP

This series of experiments shows the value of the knowledge gained froma GHBP assay, and naturally follows from the above rat experiments. Onthe basis of the prolonged half-line of the E. coli--derived GH plusGHBP complex in blood, the GHBP allows GH to persist in the blood forsufficient time to allow less frequent GH injections when the GH iscoupled to GHBP.

In 2 separate studies we inject hGH by itself, or combined andco-injected with hGHBP, in GH deficient dwarf rats. In the first studyhGH or hGH plus hGHBP are given daily or every 2 or 4 days for 8 days.The second study is designed so that hGH or hGH plus hGHBP are givendaily or every 3 or 6 days for 18 days. In both studies hGHBP givesgreater growth responses than hGH alone no matter the injectioninterval. These studies show that when hGH is injected with hGHBP theinjection frequency can be greatly reduced, to one or twice a week,without reducing the size of the growth response.

Female dwarf rats (Study A, 12-15 weeks of age, 110-130 g; Study B,50-70 days of age, 95-110 g) are randomized into groups of 8 (Study A)or 7 (Study B), and injected i.p. with tetracycline as an intravitalmarker of bone growth. All injections of GH or GHBP are givensubcutaneously in a volume of 100 microliters of solution. In eachstudy, all rats are given a daily injection of either excipient or testcompound and weighed daily.

The hGH used in rhGH (Genentech Lot N9267AX, G042A) dissolved in sterilewater. The GHBP used is produced in E. coli and purified. In this casethe GHBP has an altered sequence to the GHBP used above, the moleculebeing produced by removing the exon 3 coding domain giving a 1-5,27-238, peptide sequence.

In the first experiment the hGH and hGHBP are prepared to be injected ina volume of 0.1 ml as:

a) hGH 0.25 mg/ml or

b) hGH 1 mg/ml

c) hGH 1 mg/ml)+hGHBP (2 mg/ml)

The 8 regimes of injection are:

1) Daily injections of a), b), and c),

2) Injections every 2 days of b) and c)

3) Injections every 4 days of b) and c)

4) Injections of excipient every day.

Therefore in this design the animals injected s.c. every 2nd day receiveonly half the GH dose of the animals given daily injections and the ratsinjected every 4th day receive only a quarter of the cumulative GH dose.

In the second experiment the hGH is prepared as:

a) 0.33 mg/ml,

b) 1 mg/ml, or

c) 2 mg/ml.

The hGH+hGHBP solutions are 0.33, 1 or 2 mg/ml of hGH combined with2-fold more hGHBP (0.66, 2 or 4 mg/ml, respectively). The 9 regimes allinjected in 0.1 ml s.c. are:

1) Excipient control

2) Daily hGH injections 33 micrograms/day

3) Daily hGH injections 100 micrograms/day

4) Daily hGH injections 33 micrograms/day+66 μg of GHBP

5) Daily hGH injections 100 micrograms/day+200 μg of GHBP

6) Every 3rd day hGH injections 100 micrograms/shot

7) Every 6th day hGH injections 200 micrograms/shot

8) Every 3rd day injections 100 micrograms hGH+200 μg GHBP/shot

9) Every 6th day injections 200 micrograms hGH+400 μg GHBP/shot

Therefore in this design the animals injected s.c. every 3rd or 6th dayreceive the same cumulative GH dose (0.33 mg/kg/day) as those injecteds.c. with the low dose of GH.

The response to hGH is increased at all frequencies of injection bycombining hGHBP with the hGH. It is surprising that in study A despitedecreasing the injection frequency from daily to every 2 days, andthereby reducing the cumulative hGH dose by half, the weight gainresponse GHBP+GH injections is the same. For hGH injections the weightgain response is markedly reduced when the injections are given every 2or 4 days. The weight gain in response to eight daily injections of 0.25mg/kg hGH is identical to that for only 2 injections of the samecumulative dose of hGH given every 4 days.

In the second Experiment, two doses of hGH are given daily with orwithout a 2-fold excess of hGHBP. The response to hGH is greatlyincreased by combining hGHBP with the hGH (see below). (In a subsequentstudy in the dwarf rat the maximal weight gain response to hGH wasincreased when the hGH was complex with and injected daily s.c. withhGHBP.) As in the previous experiments GHBP improved the IGF-1 responseto the GH. This appears to be due to the GHBP-GH complex causing apreferential and disproportionate growth of the liver, an activitylacking when GH is delivered alone. Giving the same total dose of hGH at3 or 6 day intervals gives a poor growth response (compared to hGH givendaily, Group 2). The weight gain response to combined treatment withGH+GHBP is much greater. The effect of daily hGH alone is directlycompared with hGH+hGHBP given every 3 or 6 days. Infrequent injectionsof the hGHBP-hGH combination are as effective as daily injections ofhGH. These data clearly show that the co-administration of hGH+hGHBPallows the growth response to the hGH to be maintained with infrequentinjection regimes. Co-administration of GH+GHBP allows the intervalbetween injections to be extended to 6 days (weekly) without a loss ofactivity on longitudinal bone growth (measured by the tetracyclinelabelling technique) compared to injections of the same dose of GHinjected daily. The co-administration of GH+GHBP also allows a smallerdose of GH to be given less frequently for an equivalent growth response(injections every 2 or 3 days at 1/2 to 1/3 the dose in the rat).

    ______________________________________                                        STUDY B: Bone Growth and Weight Gain in 18 days                                               Bone Growth                                                                   (microns)       Weight Gain (g)                               Group           & SD            & SD                                          ______________________________________                                        1) Excipient    34.0    8.0     10.3   3.6                                    2) low GH       53.8    8.3     27.8   3.3                                    3) Hi GH        62.1    8.4     34.0   5.4                                    4) Low GH + GHBP                                                                              73.4    9.7     28.1   4.9                                    5) Hi GH + GHBP 95.1    6.5     47.0   7.4                                    6) GH/3 days    37.7    10.3    15.2   3.7                                    7) GH/6 days    36.5    11.7    16.8   6.2                                    8) GH + GHBP/3 days                                                                           56.9    15.8    28.0   6.4                                    9) GH + GHBP/6 days                                                                           47.0    4.6     18.5   4.8                                    ______________________________________                                    

In Primates

In normal juvenile Rhesus monkeys GHBP was monitored afteradministration of GHBP+hGH or after hGH alone. The somatogenic andanabolic response was determined by measuring IGF-1 concentrations. Themonkeys received either hGH daily or hGHBP+hGH weekly, and there was anexcipient injected control group. The results, primarily from bloodIGF-1 concentrations, shows that GHBP enhances the biologically activityof GH in primates. The dose of hGH (administered with twice the molarratio of hGHBP) was 0.35 mg/kg injected weekly, the doses of hGHinjected daily were 0.05 mg/kg or 0.35 mg/kg. Serum analyses indicatedthat the maximum IFG-1 response to hGH+hGHBP is greater than for hGHalong at either dosage. As measured, the hGH serum life whenadministered in combination with hGHBP was increased 2.2 times over thatof hGH administered alone. The administration of 0.35 mg/kg hGH dailystimulated serum IGF-1 less than a single weekly administration of hGH(0.35 mg/kg) complexed with hGHBP at a 2:1 molar ratio. Weeklyadministration of hGH+hGHBP (1:2 molar ratio, given subcutaneously as abolus), resulted in a physiological response greater than daily hGHadministration of the same hGH dose, or a seven-fold greater total dose.Therefore, lesser amounts of hGH can be administered and less frequentinjections given if hGH is complexed with hGHBP. In summary, in the ratand in the monkey, GHBP enhances GH activity, so that in humans asimilar enhancement is expected.

The mechanism of the above effects of the GHBP on body growth could beexplained by the greatly delayed absorption of the GH-GHBP complex froms.c. injections and then the delayed clearance from the blood of theGH-GHBP complex. Both these effects are demonstrated. The magnitude ofthis effect is quite surprising as the absorption of free GHBP wassimilar to that for GH alone. These pharmacokinetic mechanisms explain alarge part of the increased activity, and the ability to give lessfrequent injections of the GHBP+GH complex. These discoveries aresurprising as there is no prior art showing that increasing thehalf-like of GH in the blood would inevitably lead to an increasedactivity. If a molecule is retained in the blood its access to tissueswill be limited yet degradation of the molecule in the blood willcontinue. If the GH is bound to the GHBP access of the GH to a cellularGH receptor would be expected to be modified. It is clear that for themolecule to be active on tissues it must pass from the bloodstream intothe tissues, so that there are limits to the degree of delayed clearancethat is desirable.

EXAMPLE 6 Analysis of 24-hour Plasma Profiles of GHBP,GH/GH-GHBP-complex and GH in Healthy Children

We have used the LIFA to measure GHBP levels in plasma profiles fromhealthy children. GH was measured by IRMA. Fifteen 24 h plasma profilesfrom 12 healthy children (3 girls and 9 boys) of different ages (6-17years), heights (-3.7 to +3.5 SDS) and pubertal stages (1 to 4) wereexamined. Blood was withdrawn continuously for 24 h and collected in 20min fractions. Time series for GH, GHBP and GH/GHBP-complex wereanalyzed by cross-section and Fourier analysis. GH was secreted in apulsatile fashion in all subjects. The concentration of theGH/GHBP-complex varied during the sampling period, and the changescorrelated significantly with the GH pulses with correlationcoefficients reaching maximum at zero time lag. In contrast, the changesin the total GHBP concentration were minor (CV˜10%), and not correlatedto GH pulses. Fourier analysis showed similar spectral power patternsfor GH and GH/GHBP-complex, suggesting a diurnal rhythm (12-24 hperiods) as well as components of higher frequencies (around 4 hperiods). In spite of the subtle fluctuations in the total GHBPconcentration, Fourier transformation revealed a marked diurnal rhythm,while components of higher frequencies were much less abundant. Weconclude that the variations in total GHBP during a 24 h sampling periodare small and that the levels can be estimated from a single randomblood sample.

Materials and Methods

Subjects

Twelve children, 3 girls and 9 boys, of different ages (6-15 years old),heights (-3.7 to +3.5 SDS) and pubertal stages (stage 1-4), wereinvestigated at the Children's Hospital, G oteborg, Sweden. Two of thesubjects were studies on more than one occasion (Table 4). Height at thetime of the study was expressed in SD scores compared to normal Swedishchildren (Karlberg P, Taranger J, Engstrom om I, Lichtenstein H,Svennberg-Redegren I. The somatic development of children in a Swedishurban community. Acta Pediatrica Scand. 1976; Suppl. 258:1). Allchildren were healthy and well nourished, and had normal thyroid, liverand kidney function. Coeliac disease was excluded. Children withclassical GH deficiency were not included in the study. The testicularvolume was measured by orchidometer, and the pubertal stages wereclassified according to Tanner (Tanner J. M., Whitehouse R. H. Clinicallongitudinal standards for height velocity, weight velocity and stagesof puberty. (Arch. Dis. Child. 51:170-179[1976]).

                  TABLE 4                                                         ______________________________________                                        SUBJECT CHARACTERISTICS                                                       Profile                                                                             Sub-                Height                                                                              Weight                                                                              Puberty                                 No    ject   Sex     Age    (SDS) (SDS) B/T.sup.a                                                                           PH.sup.b                        ______________________________________                                        1     A      Female   6  0/12                                                                             +2.5  +2.0   1    1                               2     B      Female  12  8/12                                                                             -1.9  -2.0   1    1                               3     C      Female  13  3/12                                                                             -1.0  0      3    2                               4     D      Male     7  11/12                                                                            -0.3  +0.5   1 ml 1                               5     E      Male    11  0/12                                                                             -2.3  -2.0   2 ml 1                               6     F      Male    11  2/12                                                                             -2.0  -1.5   3 ml 1                               7     G      Male    11  3/12                                                                             +3.0  +2     2 ml 1                               8     H      Male    13  0/12                                                                             +1.0  +1.0  15 ml 3                               9     H      Male    13  9/12                                                                             +0.8  +0.8  15 ml 4                               10    I      Male    12  5/12                                                                             +0.7  +0.1   4 ml 1                               11    I      Male    12  6/12                                                                             +0.7  0      4 ml 1                               12    I      Male    13  3/12                                                                             +0.4  -0.4   5 ml 1                               13    J      Male    14  0/12                                                                             -1.8  -1.8   6 ml 1                               14    K      Male    14  7/12                                                                             -2.5  -1.5  12 ml 3                               15    L      Male    14  8/12                                                                             -3.7  -3.2   5 ml 2                               ______________________________________                                         .sup.a Breast development (B), testicular volume (T).                         .sup.b Pubic hair (PH).                                                  

Study protocol

The Children stayed at the hospital for at least 2 days. They were givena normal diet, with breakfast at 08.00 h, lunch at 12.00 h, dinner at17.00 h, and were allowed normal activity and sleep. A heparinizedneedle (Carmeda, Stockholm, Sweden) was inserted on the first evening.The following morning, at 08.00 h-09.00 h, blood withdrawal beganthrough a thrombogenic catheter (Carmeda) inserted through the needleand connected to a constant withdrawal pump (Swemed AB, G oteborg,Sweden). The rate of withdrawal was 0.5-2 ml/h and the volume of thetubing was 0.1-0.2 ml. The heparinized reservoir tubes were changedevery 20 min for 24 h, thus giving 72 samples. The blood samples werekept at room temperature and centrifuged within 24 h. Aftercentrifugation the plasma was frozen until assayed.

GH measurements

Plasma GH concentration was determined in duplicate using a polyclonalantibody-based IRMA (Pharmacia, Sweden) and the WHO First InternationalReference Preparation hGH 66217 as standard. Intra-assay variation was3.2 to 3.5% at GH levels between 2-100 mU/L. Interassay variation was5.0% and 2.7% at, GH concentrations of 10 mU/L and 40 mU/L,respectively. When appropriate, a conversion factor of 2.7 U/mg, and amolecular weight of 22 000 was used to express GH concentrations inpmol/L.

GHBP measurements

Total GHBP was measured by LIFA as previously discussed in detail.Briefly, ninety-six-well microtiter plates (Corning Glass Works,Corning, N.Y.) were coated with a monoclonal antibody directed againstGHBP (MAb 263, Agen, Australia) by incubating overnight at 4° C. with100 μL/well of antibody at 10 μg/mL in coat buffer. The coated wellswere blocked and washed. Standards (recombinant hGHBP, Genentech Inc.)or samples (50 μL per well) were dispensed into the coated wellscontaining 50 μl/well of 200 ng/ml rhGH. Plates were sealed, incubatedat room temperature for 2 h with gentle agitation, then washed beforeaddition of a monoclonal anti-hGH antibody (MAb MCB, Genentech Inc)conjugated to horseradish peroxidase (100 μl/well). After furtherincubation for 2 h at room temperature, the plates were washed six timeswith wash buffer. Freshly prepared substrate solution (0.4 g ofo-phenylenediamine dihydrochloride in one liter of PBS plus 0.4 mL of30% hydrogen peroxide) was added to the plates (100 μl per well) and theincubation carried out in the dark for 15 min at room temperature. Thereaction was stopped by the addition of 100 μl of 2.25 mol/L sulfuricacid and the absorbance at 490 nm determined. The same procedure,without the addition of rhGH to the samples, was used to measure theplasma concentration of the GH/GHBP-complex. The detection range in theLIFA was 15.6 to 1000 pmol/L. The intra- and interassay coefficients ofvariation were 7.3% and 11.3%, respectively.

Statistical analysis

The rhythmicity of the 24 h profiles was analyzed by Fouriertransformation. The original GH, GHBP and GH/GHBP-complex concentrationtime series were smoothed with a 3-point moving average (weights w₋₁=w₊₁ = 1/4, w₀ = 1/2) in order to reduce the influence of high frequencycomponents. The smoothed series were analyzed as Fourier expansions(Chatfield C. The analysis of time series. Chapman and Hall, London,1989). The results are expressed as a power spectrum, where theamplitude is plotted as a function of frequency. To analyze the data forcorrelations between GH, GHBP and GH/GHBP-complex, cross-correlationfollowed by Box-Jenkins autoregressive modeling (Haugh L, Box GEP.Identification of dynamic regression (distributed lag) models connectingtwo time series. Journal of the American Statistics Association. 1977;72:121-130) was used.

Plasma profiles of GH GH/GHBP-complex and total GHBP;

Twenty-four hour plasma profiles of GH, GH/GHBP-complex and total GHBPfrom one representative subject (profile #15) are shown in FIG. 7A.Plasma concentrations of both GH (top panel) and GH/GHBP-complex (middlepanel) varied over a wide range during the sampling period, and thechanges appeared to be synchronized in time. In contrast, the totalconcentration of GHBP (lower panel) was much less variable, and did notseem to be influenced by the changes in GH and GH/GHBP-complexconcentration.

Plasma concentrations of GH, GHBP and GH/GHBP-complex

Plasma concentrations of GH, GHBP and GH/GHBP-complex are shown in Table5. The values given are the mean and coefficients of variation (CV) foreach individual 24 h profile as well as the group average. The meansconcentration of GHBP, GH/GHBP-complex and GH varied among differentindividuals (range 109-247 pmol/L, 13-109 pmol/L and 32-215 pmol/L forGHBP, GH/GHBP-complex and GH, respectively). The highly pulsatile natureof GH and GH/GHBP-complex plasma profiles were reflected in their highCV (156.0% and 47.2%, respectively). The percentage of GH that was boundto GHBP in high GH peaks (250 pmol/L) was 14.6%. The concentration oftotal GHBP was much less variable during the sampling period, asillustrated by the low coefficient of variation (9.6%) (Table 5). Thecomplete 24 h profiles of total GHBP concentration from all subjectsillustrated that levels vary among individuals and that theconcentration for each subject is relatively constant throughout theday.

                                      TABLE 5                                     __________________________________________________________________________    Mean plasma concentration of total GHBP, GH/GHBP-complex and GH.                     Total                                                                              GHBP GH/GHBP                                                      complex                      GH   P                                                  Mean CV   Mean  CV    Mean CV                                          Profile No                                                                           (pmol/L)                                                                           (%)  (pmol/L)                                                                            (%)   (pmol/L)                                                                           (%)                                         __________________________________________________________________________    1      109.3                                                                              9.4  34.8  25.6  73.1 129.6                                       2      235.2                                                                              9.5  12.9  113.9 83.3 178.5                                       3      179.0                                                                              12.7 26.2  111.0 99.8 128.9                                       4      148.5                                                                              8.1  24.1  25.8  40.4 107.7                                       5      198.9                                                                              7.8  47.3  51.0  167.9                                                                              176.7                                       6      160.1                                                                              8.6  25.7  28.6  36.6 160.7                                       7      215.0                                                                              5.5  39.8  22.5  31.6 151.0                                       8      274.4                                                                              7.7  108.8 15.3  215.2                                                                              121.6                                       9      217.5                                                                              9.3  80.1  17.2  189.1                                                                              133.7                                       10     126.2                                                                              11.8 28.2  46.4  163.4                                                                              151.3                                       11     120.4                                                                              13.8 20.7  37.0  101.9                                                                              125.7                                       12     168.7                                                                              9.2  34.3  43.4  99.8 119.4                                       13     130.0                                                                              11.1 29.5  41.5  140.9                                                                              154.9                                       14     191.1                                                                              11.8 19.8  107.5 103.7                                                                              99.1                                        15     199.8                                                                              7.0  45.4  21.9  39.3 153.5                                       Mean:  176.5                                                                              9.6 ± 0.6                                                                       38.5  47.2 ± 9.2                                                                       110.0                                                                              139 ± 6.3                                __________________________________________________________________________

Discussion

The objective in monitoring GHBP was to investigate the possible diurnalvariations in the plasma concentration of GHBP in healthy children, andto determine if fluctuations in GHBP concentrations were correlated withthe episodic release of GH. The subjects included in our study differedin age, sex, pubertal stage, height and GH levels. From a practicalpoint of view we believe that a most useful discovery from the data isthat total GHBP concentration shows only minor variations during a 24 hsampling period, implying that a single blood sample should give a goodestimation of the total GHBP level. Nevertheless, rigorous analysis ofthe data revealed that the small (CV-10%) fluctuations in total GHBPplasma concentration account for a significant circadian rhythm. Nosignificant cross-correlation between GH pulses and changes in totalGHBP concentration was found. In contrast, the plasma concentration ofGH/GHBP-complex showed rapid fluctuations, which were highly correlatedwith the changes in GH concentration. Fourier analysis showed that theplasma patterns of both GH and GH/GHBP-complex follow a diurnal rhythmbut also possess components of higher frequencies (around 4 h periods).

The described LIFA, which was used for the GHBP measurements has theadvantages that only functional GHBP is detected and that endogenous GH,which fluctuates rapidly over a wide range, does not affect themeasurement of total GHBP concentrations. The assay can also measure theconcentration of the GH/GHBP-complex. It was recently reported that whenthe ratio of GHBP to GH exceeds 1:1, trimers can form consisting of oneGH molecule and two GHBP molecules. The trimer can not be bound by thecapture antibody, so the GH/GHBP-complex that is measured in the LIFA isthe heterodimer formed by one GH molecule and one GHBP molecule. When GHconcentration increases, (e.g. when rhGH is added to the sample in theLIFA of during endogenous GH peaks), the trimer [GH-(GHBP)2] dissociatesand dimers [GH-GBP] are formed, which can be bound by the MAb 263. Thisimplies that all the GHBP, including GHBP molecules in endogenouslyformed trimers, are detectable in the assay for the total GHBPconcentration.

We found that about 15% of GH in high peaks (>250 pmol/L) appear to bebound in the GH/GHBP-complex, but the total bound fraction of GH may behigher since some GH may have formed trimers with the GHBP.

The variation in the plasma concentration of GHBP in serial samples haspreviously been addressed in two studies (Snow, K. J., Shaw M. A., WinerL. M., Baumann G. Diurnal pattern of plasma growth hormone-bindingprotein in man. J Clin Endocrinol Metab.; 70:417-420 (1980); HochbergZ., Amit T., Zadik, Z. Twenty-four-hour profile of plasma growthhormone-binding protein. J. Clin Endocrinol Metab. 72:236-239 (1991)).The study of Snow et al., which was carried out in adults with low GHlevels, agrees with our results that there is no major variation intotal GHBP levels during the sampling period. However, since GH pulseswere absent or very low in the study by Snow et al., the possibilitythat there could be GH induced variation in GHBP levels in subjects withpulsatile GH secretion could not be excluded. In another study, byHochberg et al., GH and GHBP levels were measured in samples obtainedfrom normal children with pulsatile GH secretion and it was concludedthat within 30 min the majority of the GH pulses were accompanied byGHBP pulses. This is in contrast to the present results, where onlyminor changes in the total GHBP concentration were detected and theywere not correlated with the GH pulses. The reason for the discrepancybetween the two studies is not clear, but may be due to differencesbetween the GHBP assays. The LIFA directly measures total GHBP (i.e. thesum of free GHBP and GH-bound GHBP), while the assay used by Hochberg etal., is based on the binding of radiolabeled hGH to the GHBP and thevalues are then corrected for interference by endogenous GH. Since ourdata regarding the GH/GHBP-complex indicate that the complex is formedand cleared rapidly, it is possible that the apparent GHBP pulses, whichHochberg et al observed 30 min after a GH pulse may reflect thedesaturation of the GHBP, thereby allowing more labeled GH to be bound.

The total GHBP levels varied over a wide range in different subjects(109-247 pmol/L) and it is probably that these levels are correlated todifferences in age, sex, pubertal stage, growth velocity, etc. Weconclude that GHBP levels are relatively constant throughout the day anda single or pooled blood sample should be sufficient to estimate totalGHBP concentration. This finding should facilitate comparisons withlarger populations and illustrates the value of GHBP measurement as adiagnostic tool.

EXAMPLE GHBP Determinations of Normal and Short Stature Children

Growth hormone binding protein was assayed using the LIFA assay forhuman GHBP. The results are described in Tables 6, 7, 8 and 9 below.These tables contain summary statistics on GHBP levels in normalchildren (Table 6) as well as in children with short stature due tothree different etiologies: idiopathic growth hormone deficiency (GHD)(Table 7), (ISS) (Tale 8) and Turner syndrome (Table 9). Normal datawere obtained from samples in Genentech's control and fromcollaborations with outside investigators. The samples from childrenwith short stature were obtained as part of an ongoing post-marketingsurveillance project from Protropin® human growth hormone, the GenentechNational Cooperative Growth Study (NCGS). Now ISS children are nowlonger ideopathic in that the GHBP deficiency likely reflects anunderlying growth hormone receptior deficiency.

The summary statistics for normal children are presented by sex and ageand represent our best estimate of a normal range for GHBP. Thesestatistics consist of the mean and mean plus or minus 2 standarddeviations (SD) and were determined from the logged (base 10) values ofthe GHBP levels, then converted back to the original units. Sample sizesused in the estimates are included with the summary statistics. Notethat there were sufficient data to perform these calculations for maleand female children aged, 3, 4, and 6 through 15 only. Data fromchildren of other ages and from adults were too sparse to allow a goodestimate of the mean.

The summary statistics listed for each of the etiologies of shortstature are the sample size, means and mean plus or minus one SD. Thesevalues were computed in the same manner as that described for thenormals. The statistics are printed by age and sex for all availabledata regardless of sample size. The units of GHBP are in pmole/liter.

                  TABLE 6                                                         ______________________________________                                        Growth Hormone Binding Protein Norms                                          (GHBP Normal Range)                                                                                  Mean           Mean                                    Sex     Age      N     -2 SD    Mean  +2 SD                                   ______________________________________                                        Male     3       20    57.4     127.3 282.5                                   Male     4       21    64.6     120.2 223.5                                   Male     6       31    56.5     111.6 220.6                                   Male     7       31    78.2     143.0 261.7                                   Male     8       34    62.7     178.5 507.7                                   Male     9       36    64.9     198.1 604.7                                   Male    10       37    62.5     226.9 822.8                                   Male    11       40    70.7     234.6 779.0                                   Male    12       48    79 4     238.0 713.3                                   Male    13       33    72.5     231.7 739.9                                   Male    14       37    67.6     97.7  578.4                                   Male    15       33    51.7     173.4 581.8                                   Female   3       15    77.4     149.3 288.0                                   Female   4       17    62.0     179.3 518.6                                   Female   6       33    57.8     142.7 351.9                                   Female   7       32    73.5     175.2 417.7                                   Female   8       33    93.7     230.9 568.8                                   Female   9       36    90.8     215.7 512.4                                   Female  10       32    71.2     244.6 841.0                                   Female  11       33    97.5     285.8 838.3                                   Female  12       36    87.7     228.7 596.8                                   Female  13       36    113.0    305.9 827.8                                   Female  14       35    99.7     260.2 678.7                                   Female  15       28    122.4    345.8 976.5                                   ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        Growth Hormone Binding Protein Norms                                          (Idiopathic GHD: GHBP Levels)                                                                         Mean          Mean                                    Sex     Age      N      -2 SD   Mean  +2 SD                                   ______________________________________                                        Male    1        1      --      73.5  --                                      Male    3        3      40.3    63.7  100.7                                   Male    4        5      56.3    95.4  161.7                                   Male    5        3      79.5    96.5  117.1                                   Male    6        7      76.9    97.3  123.1                                   Male    7        1      --      59.4  --                                      Male    8        4      87.3    127.8 187.1                                   Male    9        5      81.9    128.9 203.0                                   Male    10       12     73.5    150.3 307.2                                   Male    11       9      108.5   177.5 290.2                                   Male    12       14     106.8   193.5 350.4                                   Male    13       16     102.1   164.4 264.6                                   Male    14       17     83.0    149.8 270.4                                   Male    15       17     122.4   191.1 298.3                                   Male    16       5      132.1   231.4 405.3                                   Male    17       4      136.0   165.0 200.2                                   Male    18       1      --      772.5 --                                      Female  3        1      --      55.8  --                                      Female  5        2      77.4    82.5  87.9                                    Female  6        3      117.8   157.6 210.8                                   Female  8        2      200.3   241.3 290.7                                   Female  9        2      43.8    59.7  81.3                                    Female  10       3      83.4    253.9 772.7                                   Female  1        10     91.0    162.5 290.3                                   Female  2        7      89.0    189.9 405.2                                   Female  13       2      86.6    154.5 275.9                                   Female  5        2      294.0   398.6 540.4                                   ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Growth Hormone Binding Protein Norms                                          (Turner Syndrome: GHBP Levels)                                                                        Mean          Mean                                    Sex     Age      N      -2 SD   Mean  +2 SD                                   ______________________________________                                        Female   4       2      93.2    118.1 149.5                                   Female   5       4      84.1    108.3 149.4                                   Female   6       7      65.3    135.8 282.2                                   Female   7       4      97.5    146.1 218.9                                   Female   8       5      116.3   194.5 325.4                                   Female   9       10     107.9   199.0 367.2                                   Female  10       11     105.8   182.7 315.7                                   Female  11       8      181.0   241.9 323.2                                   Female  12       8      139.4   286.4 588.2                                   Female   3       9      133.3   241.8 438.7                                   Female  14       15     222.2   321.4 464.8                                   Female  15       6      101.0   189.0 353.5                                   Female  16       6      147.4   237.6 383.0                                   Female  17       2      117.6   136.0 157.4                                   ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        Growth Hormone Binding Protein Norms                                          (Idiopathic Short Stature: GHBP Levels)                                                               Mean          Mean                                    Sex     Age      N      -2 SD   Mean  +2 SD                                   ______________________________________                                        Male     2       1      --      37.3  --                                      Male     3       8      46.0    100.2 218.5                                   Male     4       14     68.6    96.3  135.4                                   Male     5       24     61.0    86.1  121.6                                   Male     6       17     47.9    65.1  88.4                                    Male     7       35     52.9    83.0  130.0                                   Male     8       29     61.1    90.8  135.0                                   Male     9       25     64.7    110.7 189.3                                   Male    10       45     69.5    108.3 168.6                                   Male    11       43     67.0    106.1 168.0                                   Male    12       77     70.5    109.5 170.2                                   Male    13       73     73.5    117.9 189.3                                   Male    14       71     70.8    110.4 172.0                                   Male    15       51     64.7    113.8 200.3                                   Male    16       12     55.9    102.9 189.4                                   Male    17       5      80.3    112.7 158.2                                   Male    18       2      64.5    195.0 589.6                                   Male    19       1      --      93.8  --                                      Male    21       1      --      184.3 --                                      Male    23       1      --      36.9  --                                      Female   1       1      --      63.3  --                                      Female   2       2      56.8    57.4  58.0                                    Female   3       2      45.3    88.2  171.4                                   Female   4       2      87.1    88.0  89.0                                    Female   5       4      50.8    91.4  164.5                                   Female   6       5      45.6    84.7  157.4                                   Female   7       6      76.6    109.2 155.6                                   Female   8       9      75.4    116.0 178.6                                   Female   9       9      89.8    120.7 162.2                                   Female  10       19     102.1   174.9 299.7                                   Female  11       31     84.3    139.3 230.1                                   Female  12       20     89.4    150.1 252.2                                   Female  13       17     110.1   146.3 194.3                                   Female  14       9      102.1   160.9 253.7                                   Female  15       4      56.5    110.7 216.8                                   Female  16       2      148.1   182.2 224.1                                   ______________________________________                                    

FIG. 8A-8E graphically illustrates the difference between normal GHBPand various etiologies. Plotted are GH binding protein levels from theNational Cooperative Growth Study patients (log concentration of GHBP vsthe age of patient). The crossbars represent mean values; solid verticallines are plus or minus 1 SDs; dotted vertical lines are plus or minustwo SDs. The separate black dots each represent one patient. (8A)Idiopathic GHD for males; (8B) Idiopathic GHD for females; (8C)Idiopathic short stature for males; (8D) Idiopathic short stature forfemales; (8E) Turner Syndrome.

The clinical utility of the LIFA assay for distinguishing betweennormals and various etiologies can be seen in FIG. 8(A-E). This isparticularly pronounced in FIG. 8C and D where the idiopathic shortstature patients are predominantly below the mean for normal in bothmales and females. This provides a clinically valuable diagnostic testfor evaluating the level of GHBP, and indirectly a presumptivedetermination of growth hormone receptor.

Ideopathic short stature is no longer ideopathic in that it can now beviewed as a state of relative GH resistance. Based upon the relativelack of GHBP this resistance is likely due to a deficiency in GHreceptors that are capable of responding to GH. Current therapy of ISSinvolves daily hGH injections. One approach to the deficiency in GHreceptors is to administer higher doses of GH to stimulate thosereceptors that are present. A dosage of from 1.1 to 10 times that nowused is expected to increase the GH response. Alternatively, now thatthe underlying basis of ISS is revealed, new treatment options differentfrom that currently used are suggested. In GH deficiency and Turner'ssyndrome, situations of normal GHBP and presumably normal GH receptorresponsiveness, GHBP+GH is the logical treatment. In ISS patients,GHBP+GH may also be used to maximize the response by those GH receptorspresent. However, in ISS, IGF-1 therapeutic treatment is also indicated.IGF-1 is given alone or with IGF binding protein it may becoadministered with GH, and/or with GHBP. Since ISS patients havereduced GHBP, the GHBP+GH combination elevates the response by those GHreceptors present. Administration of therapeutic amounts of IGF-1 orIGF-1 plus IGF BP elevates the effective serum IGF-1, thus partiallycircumventing the defective GH response and stimulating IGF-1 dependentresponses.

What is claimed is:
 1. A ligand mediated immunofunctional method fordetermining the amount of a polypeptide hormone binding protein selectedfrom the group consisting of a growth hormone binding protein factor, anepidermal growth factor binding protein, an insulin-like growth factor(IGF) binding protein, a platelet derived growth factor (PDGF) bindingprotein, a nerve growth factor (NGF) finding protein, an insulin bindingprotein, a corticotropin releasing factor (CRF) binding protein, atransforming growth factor beta (TGF-β) binding protein and an activinbinding protein in a liquid sample comprising:(a) contacting said liquidsample with: 1) a first antibody attached to a solid phase carrier,wherein said first antibody is specific for epitopes on said polypeptidehormone binding protein such that in the presence of antibody thehormone binding sites remain available on the binding protein forbinding to the ligand polypeptide hormone, thereby forming a complexbetween said first antibody and said polypeptide hormone bindingprotein; and 2) said ligand polypeptide hormone for a period of timesufficient to saturate all available ligand hormone binding sites onsaid polypeptide hormone binding protein thereby forming a saturatedcomplex; (b) contacting said saturated complex with a detectably labeledsecond antibody which is specific for epitopes on said ligandpolypeptide hormone which are available for binding when said ligandpolypeptide hormone is bound to said polypeptide hormone bindingprotein; and (c) quantitatively analyzing the amount of said labeledsecond antibody bound as a measure of said polypeptide hormone bindingprotein in said liquid sample.
 2. The method of claim 1 wherein step (b)ligand polypeptide hormone is selected from the following: a growthhormone, an epidermal growth factor, an insulin-like growth factor-1, aplatelet derived growth factor, a nerve growth factor and activin, or afragment of such polypeptide hormone having binding affinity for saidpolypeptide hormone binding protein.
 3. A mouse hybridoma producingantibody specific for human growth hormone designated as the line HGH-Bwith ATCC number HB-10596.
 4. Antibody produced by the mouse hybridomaHGH-B, ATCC number HB-10596.
 5. The method of claim 1 wherein saidpolypeptide hormone binding protein is human growth hormone bindingprotein, said ligand polypeptide hormone is human growth hormone andsaid liquid sample is from a patient suspected of a clinical conditionselected from Laron dwarfism, idiopathic short stature, Turnerssyndrome, growth hormone binding protein deficiency, growth hormonereceptor deficiency, growth hormone binding protein excess, growthhormone receptor excess, autoimmune growth disorders and growth hormonedeficiency.
 6. The method of claim 1 wherein said polypeptide hormonebinding protein is human growth hormone binding protein, said ligandpolypeptide hormone is human growth hormone and said liquid sample is afluid from a patient suspected of a clinical condition selected fromLaron dwarfism, idiopathic short stature, Turners syndrome, growthhormone binding protein deficiency, growth hormone receptor deficiencyand growth hormone deficiency, wherein said fluid is selected from thegroup consisting of serum, plasma, lymph fluid, synovial fluid,follicular fluid, seminal fluid, amniotic fluid, milk, whole blood,urine, spinal fluid, saliva, sputum, tears, perspiration and mucus. 7.In a method of assaying for the presence and amount of human growthhormone binding protein in a liquid sample using a modified immunometricassay utilizing monoclonal antibody, the improvement comprising:(a)attaching the human growth hormone binding protein to a solid phaseusing antibody specific for epitopes on said human growth hormonebinding protein such that the hormone binding sites remain available forbinding to human growth hormone, thereby forming a complex between saidantibody and said human growth hormone binding protein; (b) saturatingthe complex of step (a) with human growth hormone for a period of timesufficient to saturate all available human growth hormone binding siteson said human growth hormone binding protein thereby forming a saturatedcomplex; (c) contacting said saturated complex with a detectably labeledsecond antibody which is specific for epitopes on human growth hormonewhich are available for binding when human growth hormone is bound tosaid human growth hormone binding protein; and (d) quantitativelyanalyzing the amount of said labeled second antibody bound as a measureof said human growth hormone binding protein in said liquid sample;wherein said liquid sample is from a patient suspected of a clinicalcondition selected from Laron dwarfism, idiopathic short stature,Turners syndrome, growth hormone binding protein deficiency, growthhormone receptor deficiency and growth hormone deficiency.